What Is Bioreactor?

A bioreactor is a vessel in which is carried out a chemical process which involves organisms or biochemically active substances derived from such organisms.

Bioreactors are commonly cylindrical, ranging in size from some liter to cube meters,and are often made of stainless steel.

Bioreactor design is quite a complex engineering task. Under optimum conditions the microorganisms or cells will reproduce at an astounding rate. The vessel's environmental conditions like gas (i.e., air, oxygen, nitrogen, carbon dioxide) flowrates, temperature, pH and dissolved oxygen levels, and agitation speed need to be closely monitored and controlled. One bioreactor manufacturer, Broadley-James Corporation, uses vessels, sensors, controllers, and a control system, digitally networked together for their bioreactor system.

Continuous flow stirred tank reactors (chemostat) In the continuous flow, stirred tank reactor (CSTR or chemostat) fresh medium is fed into the bioreactor at a constant rate, and medium mixed with cells leaves the bioreactor at the same rate. A fixed bioreactor volume is maintained and ideally, the effluent stream should have the same composition as the bioreactor contents. The culture is fed with fresh medium containing one and sometimes two growth-limiting nutrients such as glucose. The concentration of the cells in the bioreactor is controlled by the concentration of the growth-limiting nutrient. A steady state cell concentration is reached where the cell density and substrate concentration are constant. The cell growth rate (µ) is controlled by the dilution rate (D) of growth limiting nutrient.

Cell culture bioreactors are categorized into two types: 1. Those that are used for cultivation of anchorage dependent cells (e.g. primary cultures derived from normal tissues and diploid cell lines. 2. Those that are used for the cultivation of suspended mammalian cells (e.g. cell lines derived from cancerous tissues and tumors, transformed diploid cell lines, hybridomas). In some cases the bioreactor may be modified to grow both anchorage dependent and suspended cells. Ideally any cell culture bioreactor must maintain a sterile culture of cells in medium conditions which maximize cell growth and productivity.

Fouling can harm the overall sterility and efficiency of the bioreactor, especially the heat exchangers. To avoid it the bioreactor must be easily cleanable and must be as smooth as possible (therefore the round shape).

Heat exchange is needed to maintain the bioprocess at a constant temperature. Biological fermentation is a major source of heat, therefore in most cases bioreactors need water refrigeration. They can be refrigerated with an external jacket or, for very large vessels, with internal coils.

Optimal oxygen transfer is perhaps the most difficult task to accomplish. Oxygen is poorly soluble in water -and even less in fermentation broths- and is relatively scarce in air (20.8%). Oxygen transfer is usually helped by agitation, that is also needed to mix nutrients and to keep the fermentation homogeneous. There are however limits to the speed of agitation, due both to high power consumption (that's proportional to the cube of the speed) and the damage to organisms due to excessive tip speed.

Bioreactor treatment may be performed using microorganisms growing in suspension in the fluid or attached on a solid growth support medium. In suspended growth systems, such as fluidized beds or sequencing batch reactors, contaminated groundwater is circulated in an aeration basin where a microbial population aerobically degrades organic matter and produces carbon dioxide, water, and biomass. The biomass is settled out in a clarifier, then either recycled back to the aeration basin or disposed of as sludge. In attached growth systems, such as upflow fixed film bioreactors, rotating biological contactors (RBCs), and trickling filters, microorganisms are grown as a biofilm on a solid growth support matrix and water contaminants are degraded as they diffuse into the biofilm. Support media include solids that have a large surface area for bacterial attachment.

A bioreactor landfill operates to rapidly transform and degrade organic waste. The increase in waste degradation and stabilization is accomplished through the addition of liquid and air to enhance microbial processes. This bioreactor concept differs from the traditional “dry tomb” municipal landfill approach.

A bioreactor landfill is not just a single design and will correspond to the operational process invoked. There are three different general types of bioreactor landfill configurations:

Aerobic - In an aerobic bioreactor landfill, leachate is removed from the bottom layer, piped to liquids storage tanks, and re-circulated into the landfill in a controlled manner. Air is injected into the waste mass, using vertical or horizontal wells, to promote aerobic activity and accelerate waste stabilization. Anaerobic - In an anaerobic bioreactor landfill, moisture is added to the waste mass in the form of re-circulated leachate and other sources to obtain optimal moisture levels. Biodegradation occurs in the absence of oxygen (anaerobically) and produces landfill gas. Landfill gas, primarily methane, can be captured to minimize greenhouse gas emissions and for energy projects. Hybrid (Aerobic-Anaerobic) - The hybrid bioreactor landfill accelerates waste degradation by employing a sequential aerobic-anaerobic treatment to rapidly degrade organics in the upper sections of the landfill and collect gas from lower sections. Operation as a hybrid results in the earlier onset of methanogenesis compared to aerobic landfills The Solid Waste Association of North America (SWANA) has defined a bioreactor landfill as "any permitted Subtitle D landfill or landfill cell where liquid or air is injected in a controlled fashion into the waste mass in order to accelerate or enhance biostabilization of the waste." The United States Environmental Protection Agency (EPA) is currently collecting information on the advantages and disadvantages of bioreactor landfills through case studies of existing landfills and additional data so that EPA can identify specific bioreactor standards or recommend operating parameters.

Features Unique to Bioreactor Landfills: The bioreactor accelerates the decomposition and stabilization of waste. At a minimum, leachate is injected into the bioreactor to stimulate the natural biodegradation process. Bioreactors often need other liquids such as stormwater, wastewater, and wastewater treatment plant sludges to supplement leachate to enhance the microbiological process by purposeful control of the moisture content and differs from a landfill that simple recirculates leachate for liquids management. Landfills that simply recirculate leachate may not necessarily operate as optimized bioreactors.

J Biomech, 2005 Mar, 38(3), 543 - 9
3-D computational modeling of media flow through scaffolds in a perfusion bioreactor; Porter B et al.; Media perfusion bioreactor systems have been developed to improve mass transport throughout three-dimensional (3-D) tissue-engineered constructs cultured in vitro . In addition to enhancing the exchange of nutrients and wastes, these systems simultaneously deliver flow-mediated shear stresses to cells seeded within the constructs . Local shear stresses are a function of media flow rate and dynamic viscosity, bioreactor configuration, and porous scaffold microarchitecture . We have used the Lattice-Boltzmann method to simulate the flow conditions within perfused cell-seeded cylindrical scaffolds . Microcomputed tomography imaging was used to define the scaffold microarchitecture for the simulations, which produce a 3-D fluid velocity field throughout the scaffold porosity . Shear stresses were estimated at various media flow rates by multiplying the symmetric part of the gradient of the velocity field by the dynamic viscosity of the cell culture media . The shear stress algorithm was validated by modeling flow between infinite parallel plates and comparing the calculated shear stress distribution to the analytical solution . Relating the simulation results to perfusion experiments, an average surface shear stress of 5x10(-5)Pa was found to correspond to increased cell proliferation, while higher shear stresses were associated with upregulation of bone marker genes . This modeling approach can be used to compare results obtained for different perfusion bioreactor systems or different scaffold microarchitectures and may allow specific shear stresses to be determined that optimize the amount, type, or distribution of in vitro tissue growth.

J Biotechnol, 2005 Mar 2, 116(1), 61 - 77 Epub 2004 Nov 30.
Pellet morphology, culture rheology and lovastatin production in cultures of Aspergillus terreus; Casas Lopez JL et al.; Pellet growth of Aspergillus terreus ATCC 20542 in submerged batch fermentations in stirred bioreactors was used to examine the effects of agitation (impeller tip speed u(t) of 1.01-2.71 ms(-1)) and aeration regimens (air or an oxygen-enriched mixture containing 80% oxygen and 20% nitrogen by volume) on the fungal pellet morphology, broth rheology and lovastatin production . The agitation speed and aeration methods used did not affect the biomass production profiles, but significantly influenced pellet morphology, broth rheology and the lovastatin titers . Pellets of approximately 1200mum initial diameter were reduced to a final stable size of approximately 900mum when the agitation intensity was >/=600rpm (u(t)>/=2.03ms(-1)) . A stable pellet diameter of approximately 2500mum could be attained in less intensely agitated cultures . These large fluffy pellets produced high lovastatin titers when aerated with oxygen-enriched gas but not with air . Much smaller pellets obtained under highly agitated conditions did not attain high lovastatin productivity even in an oxygen-enriched atmosphere . This suggests that both an upper limit on agitation intensity and a high level of dissolved oxygen are essential for attaining high titers of lovastatin . Pellet size in the bioreactor correlated equally well with the specific energy dissipation rate and the energy dissipation circulation function . The latter took into account the frequency of passage of the pellets through the high shear regions of the impellers . Pellets that gave high lovastatin titers produced highly shear thinning cultivation broths.

Appl Microbiol Biotechnol . 2005 Jan 13; {Epub ahead of print}
Oligomeric compounds formed from 2,5-xylidine (2,5-dimethylaniline) are potent enhancers of laccase production in Trametes versicolor ATCC 32745; Kollmann A et al.; Numerous chemicals, including the xenobiotic 2,5-xylidine, are known to induce laccase production in fungi . The present study was conducted to determine whether the metabolites formed from 2,5-xylidine by fungi could enhance laccase activity . We used purified laccases to transform the chemical and then we separated the metabolites, identified their chemical structure and assayed their effect on enzyme activity in liquid cultures of Trametes . versicolor . We identified 13 oligomers formed from 2,5-xylidine . (4E)-4-(2,5-dimethylphenylimino)-2,5-dimethylcyclohexa-2,5-dienone at 1.25x10(-5) M was an efficient inducer, resulting in a nine-fold increase of laccase activity after 3 days of culture . Easily synthesized in one step (67% yield), this compound could be used in fungal bioreactors to obtain a great amount of laccases for biochemical or biotechnological purposes, with a low amount of inducer.

Ann N Y Acad Sci, 2004 Nov, 1027, 85 - 98
Modeling of phosphate ion transfer to the surface of osteoblasts under normal gravity and simulated microgravity conditions; Mukundakrishnan K et al.; We have modeled the transport and accumulation of phosphate ions at the remodeling site of a trabecular bone consisting of osteoclasts and osteoblasts situated adjacent to each other in straining flows . Two such flows are considered; one corresponds to shear levels representative of trabecular bone conditions at normal gravity, the other corresponds to shear level that is representative of microgravity conditions . The latter is evaluated indirectly using a simulated microgravity environment prevailing in a rotating wall vessel bioreactor (RWV) designed by NASA . By solving the hydrodynamic equations governing the particle motion in a RWV using a direct numerical simulation (DNS) technique, the shear stress values on the surface of the microcarriers are found . In our present species transfer model, osteoclasts release phosphate ions (Pi) among other ions at bone resorption sites . Some of the ions so released are absorbed by the osteoblast, some accumulate at the osteoblast surface, and the remainder are advected away . The consumption of Pi by osteoblasts is assumed to follow Michaelis-Menten (MM) kinetics aided by a NaPi cotransporter system . MM kinetics views the NaPi cotransporter as a system for transporting extracellular Pi into the osteoblast . Our results show, for the conditions investigated here, the net accumulation of phosphate ions at the osteoblast surface under simulated microgravity conditions is higher by as much as a factor of three . Such increased accumulation may lead to enhanced apoptosis and may help explain the increased bone loss observed under microgravity conditions.

Artif Organs, 2005 Jan, 29(1), 58 - 66
A novel bioartificial liver with culture of porcine hepatocyte aggregates under simulated microgravity; Hochleitner B et al.; An extracorporeal bioartificial liver device could provide vital support to patients suffering from acute liver failure . We designed a novel, customized bioreactor for use as a bioartificial liver (patent pending) . The Innsbruck Bioartificial Liver (IBAL) contains aggregates of porcine hepatocytes grown under simulated microgravity . The culture vessel rotates around its longitudinal axis and is perfused by two independent circuits . The circuit responsible for exchange of plasma components with the patient consists of a dialysis tube winding spirally around the internal wall of the culture vessel . IBAL was evaluated in vitro . Viability tests showed sufficient viability of hepatocytes for up to 10 days . Cytologic examination of samples from the bioreactor showed liver cell aggregates . These were also examined by electron microscopy . A number of biochemical parameters were analyzed . In conclusion, cell culture is possible for at least 10 days in the IBAL system, organoid hepatocyte aggregates are formed and synthetic activity of the hepatocytes was demonstrated.

Biotechnol Bioeng . 2005 Jan 10; {Epub ahead of print}
Novel application of oxygen-transferring membranes to improve anaerobic wastewater treatment; Kappell AS et al.; Anaerobic biological wastewater treatment has numerous advantages over conventional aerobic processes; anaerobic biotechnologies, however, still have a reputation for low-quality effluents and operational instabilities . In this study, anaerobic bioreactors were augmented with an oxygen-transferring membrane to improve treatment performance . Two anaerobic bioreactors were fed a synthetic high-strength wastewater (chemical oxygen demand, or COD, of 11,000 mg l(-1)) and concurrently operated until biomass concentrations and effluent quality stabilized . Membrane aeration was then initiated in one of these bioreactors, leading to substantially improved COD removal efficiency (>95%) compared to the unaerated control bioreactor ( approximately 65%) . The membrane-augmented anaerobic bioreactor required substantially less base addition to maintain circumneutral pH and exhibited 75% lower volatile fatty acid concentrations compared to the unaerated control bioreactor . The membrane-aerated bioreactor, however, failed to improve nitrogenous removal efficiency and produced 80% less biogas than the control bioreactor . A third membrane-augmented anaerobic bioreactor was operated to investigate the impact of start-up procedure on nitrogenous pollutant removal . In this bioreactor, excellent COD (>90%) and nitrogenous (>95%) pollutant removal efficiencies were observed at an intermediate COD concentration (5,500 mg l(-1)) . Once the organic content of the influent wastewater was increased to full strength (COD = 11,000 mg l(-1)), however, nitrogenous pollutant removal stopped . This research demonstrates that partial aeration of anaerobic bioreactors using oxygen-transferring membranes is a novel approach to improve treatment performance . Additional research, however, is needed to optimize membrane surface area versus the organic loading rate to achieve the desired effluent quality . (c) 2005 Wiley Periodicals, Inc.

Yi Chuan, 2004 Nov, 26(6), 977 - 83
{The application of chloroplast genome transformation in higher plant.}; Wang YF et al.; The chloroplast genome transformation of higher plant has become the research hotspot of plant genetic engineering with several advantages over nuclear genetic engineering, and it has become into a powerful approach for both basic and applied research . This paper presents a brief summary for the principle and methods of chloroplast genome transformation in higher plant . The particular emphasis was placed on its application in the basic and applied research . These applications include the studies of rearrangement of Rubisco and the structure, transcription, translation and RNA editing of genes in chloroplast; the producing of antibodies, vaccines, poly-beta-hydroxybutyrate and bio-elastic protein using chloroplast as bioreactor; the generating of transgenic plants with resistance to insect, disease, herbicide and drought; and the decreasing the gene flow of transgenic plants.

Yi Chuan, 2004 Sep, 26(5), 567 - 73
{Mammary Gland Dual-Expression Construct Containing Prolactin and Foreign DNAs Can Promote the Expression of Foreign Proteins in Mammary Cells.}; Liu FT et al.; To investigate the feasibility in the promotion of the expression of foreign proteins, such as human lactinferrin (hLF) and thrombopoietin (TPO), in the transgenic animals-mammary gland bioreactor by bovine prolactin (bPRL), two types of mammary gland dual-expression vectors containing bPRL and foreign DNAs were constructed . In one type of vector, both foreign and bPRL DNAs were linked by internal ribosome entry site (IRES) which shared one goat 6.7 kb of beta-casein promoter; while in the other type of vector, the transcriptions of the foreign gene aswell as bPRL were respectively directed by 6.7 kb goat beta-casein promoter and 2.0 kb of goat beta-casein promoter . After transfection of the vectors with lipofectin method, the expression of foreign proteins were measured by RT-PCR as well as ELISA assay . The results showed that bPRL could obviously promote the expression of foreign proteins (hLF and TPO) in cultured cells . The hLF expression level was increased from 12.6 mug/L to 18.4 mug/L and 37.2 mug/L in the COS-7 cells (African green monkey kidney cell) (P<0.05), and from 13.7 mug/L to 20.7 mug/L and 19.9 mug/L in the HC-11 cells (epithelial cell of mouse mammary gland) (P<0.05) by two vectors, respectively . TPO expression level was increased from 572 ng/L to 1340 ng/L in the COS-7 cell (P<0.05), and from 783 ng/L to 1040 ng/L in the HC-11 cell (P<0.05) by the vector which have two promoters . This work indicate that bovine prolactin can effectively increase the expression of foreign genes in mammal cells.

Yi Chuan, 2004 Jul, 26(4), 537 - 43
{Achievements and Applications in Making Chicken Chimeras Using BCs.}; Yan HF et al.; The technology of producing chicken chimeras using blastodermal cells is very important not only in the field of transgenic chicken bioreactor but also in searching for efficient ways to conserve avian genetic resource . The basic processes for producing chicken chimeras consist of: (1) Setting up the color model; (2) Separating and dissociating of donor embryos; (3) Compromising of the recipient embryos; (4) Windowing and recovering the recipient eggs; (5) Cells injecting; (6) Method of hatching . The progress, obstacles and prospects of producing chicken chimeras via BCs were discussed in this paper.

Yi Chuan, 2003 Jan, 25(1), 107 - 12
{Plants as bioreactor for the production of pharmaceutical proteins.}; Xiao NZ et al.; Transgenic plant as bioreactor has been used to produce recombinant proteins for medicinal purposes,including mammalian antibodies,blood substitutes and vaccines.As the demand for biopharmaceuticals is expected to increase,transgenic plants have the potential to provide virtually unlimited quantities of proteins for use as tools in both human health care and the bioscience.This paper reviews the recent developments in this field and the prospect of commercial applications.

J Biotechnol, 2005 Feb 23, 115(4), 379 - 386 Epub 2004 Nov 21.
Selection of new over-producing derivatives for the improvement of extracellular lipase production by the non-conventional yeast Yarrowia lipolytica; Fickers P et al.; The non-conventional yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene . Mutant strains with enhanced productivity were previously obtained either by chemical mutagenesis or genetic engineering . In this work, we used one of these mutants, named LgX64.81 to select new overproducing strains following by amplification of the LIP2 gene . We also developed a process for lipase production in bioreactors and compared lipase production levels in batch and fed-batch cultures . Batch culture led to a lipase production of 26450Uml(-1) in a media containing olive oil and tryptone as carbon and nitrogen sources . Feeding of a combination of tryptone and olive oil at the end of the exponential growth phase yielded to lipase activity of 158246Uml(-1) after 80h of cultivation . In addition this production system developed for the extracellular lipase could also be applied for other heterologous protein production since we have demonstrated that LgX64.81 is an interesting alternative host strain.

J Biotechnol, 2005 Feb 23, 115(4), 333 - 43 Epub 2004 Nov 24.
Comparison of different fungal enzymes for bleaching high-quality paper pulps; Sigoillot C et al.; Wild and recombinant hydrolases and oxidoreductases with a potential interest for environmentally sound bleaching of high-quality paper pulp (from flax) were incorporated into a totally chlorine free (TCF) sequence that also included a peroxide stage . The ability of feruloyl esterase (from Aspergillus niger) and Mn(2+)-oxidizing peroxidases (from Phanerochaete chrysosporium and Pleurotus eryngii) to decrease the final lignin content of flax pulp was shown . Laccase from Pycnoporus cinnabarinus (without mediator) also caused a slight improvement of pulp brightness that was increased in the presence of aryl-alcohol oxidase . However, the best results were obtained when the laccase treatment was performed in the presence of a mediator, 1-hydroxybenzotriazol (HBT), enabling strong delignification of pulps . The enzymatic removal of lignin resulted in high-final brightness values that are difficult to attain by chemical bleaching of this type of pulp . A partial inactivation of laccase by HBT was observed but this negative effect was strongly reduced in the presence of pulp . The good results obtained with the same laccase expressed in A . niger at bioreactor scale, revealed the feasibility of using recombinant laccase for bleaching high-quality non-wood pulps in the presence of a mediator.

J Biotechnol, 2005 Feb 9, 115(3), 307 - 15
Efficient production of a soluble fusion protein containing human beta-defensin-2 in E . coli cell-free system; Chen H et al.; Human beta-defensin-2 (hBD2), a small cationic peptide, exhibits a broad range of antimicrobial activity and does not cause microbial resistance . In order to produce hBD2 efficiently, an Escherichia coli cell-free biosynthesis system has been developed as an alternative method . A specific plasmid pIVEX2.4c-trxA-shBD2 was constructed for the cell-free expression of fusion protein (hBD2 linked with His-Tag and Trx-Tag) . This allowed enhancement of protein stability and facilitated downstream purification process . Significant amount of target fusion protein was synthesized in the batch-mode bioreactor by optimizing the reaction conditions . About five-fold improvement of productivity (ca . 2.0mg/ml soluble fusion protein) could be achieved by using a continuous exchange cell-free (CECF) system compared to batch system . One-step affinity chromatographic process was developed to recover high purity fusion protein (95.2%) with overall recovery ratio of about 50% . The fusion protein was cleaved by cyanogens bromide (CNBr), and the mature hBD2 had demonstrated strong inhibition on the growth of E . coli D31 at low concentration.

J Biotechnol, 2005 Feb 9, 115(3), 291 - 301 Epub 2004 Nov 05.
The influence of the chemical composition of cell culture material on the growth and antibody production of hybridoma cells; Heilmann K et al.; The multiplication and antibody production of murine hybridoma cells cultured on five different polymer membranes were tested and compared with conventional tissue culture polystyrene (TCPS) . Membranes were prepared from polyacrylonitrile (PAN) and acrylonitrile copolymerized with N-vinylpyrrolidone (NVP20, NVP30), Na-methallylsulfonate (NaMAS) and N-(3-amino-propyl-methacrylamide-hydrochloride) (APMA) . Cell number and antibody concentration were quantified as criteria for viability and productivity . Adhesion of hybridoma cells was characterized by vital and scanning electron microscopy . The results suggest that a strong adhesion of cells, observed on APMA and TCPS, increased cell growth but reduced monoclonal antibody production . In contrast membranes with lowered adhesivity such as NVP20 provided favourable conditions for monoclonal antibody production . In addition it was shown that this membrane also possessed a minor fouling as indicated by the low decrease of water flux across the membrane after protein adsorption . It was concluded that NVP20 could be a suitable material for the development of hollow fibre membranes for bioreactors.

Protein Pept Lett, 2005 Jan, 12(1), 85 - 8
Production of minichaperone (sht GroEL191-345) and its function in the refolding of recombinant human interferon gamma; Guan YX et al.; The recombinant minichaperone sht GroEL191-345 was cultivated in a 3.7 L stirred bioreactor with the high yield of 216.2 mg/L broth . In the refolding of recombinant human interferon gamma (rhuIFN-gamma) inclusion bodies, more than 2-3 fold enhancement in protein mass recovery and total activity were observed in the presence of free or immobilized minichaperone to the refolding buffer.

Res Microbiol, 2005 Jan-Feb, 156(1), 82 - 7
Optimisation of growth conditions for continuous culture of the hyperthermophilic archaeon Thermococcus hydrothermalis and development of sulphur-free defined and minimal media; Postec A et al.; The hyperthermophilic archaeon Thermococcus hydrothermalis was cultivated in continuous culture in a gas-lift bioreactor in the absence of elemental sulphur on both proteinaceous and maltose-containing media . Optimal conditions (pH, temperature and gas flow rate), determined on complex media that yielded maximal growth rate and maximal steady state cell density, were obtained at 80 degrees C, pH 6 and gas sparging at 0.2 v v(-1) min(-1) . Higher steady state cell densities were obtained on a medium containing maltose and yeast extract . In order to design a defined and minimal media, the nutritional requirements of T . hydrothermalis were then investigated using continuous culture in the absence of elemental sulphur in the gas-lift bioreactor . First, the complex nutriments were replaced and a defined medium containing maltose, 19 amino acids and the two nitrogenous bases adenine and thymine, was determined . Secondly, selective feedings and withdrawal of amino acids showed requirements for 14 amino acids.

Biotechnol Appl Biochem . 2005 Jan 6; {Epub ahead of print}
Application of radial-flow bioreactor on production of beta1, 3-N-acetylglucosaminyltransferase-2 fused with GFP uv using stably transformed insect cell lines; Kwon MS et al.; A radial-flow bioreactor (RFB) with a reactor volume of 5 ml was applied to produce human beta1, 3-N-acetylglucosaminyltransferase (beta3GnT) using two stably transformed insect cell lines . When air was supplied to the RFB, cell growth was stopped at 4-d culture and beta3GnT was not detected . However, with a supply of pure oxygen, the cell concentration, assumed from glucose consumption, increased 1.3 x 10 7 cells/ml . Insect cells attached to polyvinyl alcohol (PVA) matrixes packed in the RFB and grew confluently . The 5.6 mU/ml of beta3GnT was produced under the conditions of a pure oxygen supply and the addition of glucose and glutamine . This RFB was first applied on the beta3GnT production using stably transformed insect cells . The amount of beta3GnT production in only a 5 ml scale RFB was comparable to that of a 100 ml shaking flask culture.

Int J Artif Organs, 2004 Nov, 27(11), 962 - 70
Hydrodynamic stimulation and long term cultivation of nucleus pulposus cells: a new bioreactor system to induce extracellular matrix synthesis by nucleus pulposus cells dependent on intermittent hydrostatic pressure; Gokorsch S et al.; A novel bioreactor system was constructed to induce extracellular matrix (ECM) synthesis by intervertebral disc (ID) cells due to intermittent hydrostatic pressure . The developed system is completely sterilizable and reusable . It is viable for cultivation, immobilization, and stimulation of various other cell types and tissues especially for cartilage . The custom made lid allows long-run cultivation through semi-continuous operation . Manual interferences and therefore the risk of contamination are reduced . Sampling, medium changing and addition of supplements are easily performed from the connected conditioning vessel, which could be placed in an incubator . For the present investigations nucleus pulposus cells from pigs were taken and immobilized in agarose to obtain three-dimensional cell matrix constructs which were subjected to intermittent hydrostatic pressure . Afterwards the construct was biochemically examined . The proven constituents of ECM were found to be released in dependence of the magnitude and profile of the applied pressure.

Biotechnol Bioeng . 2005 Jan 5; {Epub ahead of print}
Relationships between hydrodynamics and rheology of flocculating yeast suspensions in a high-cell-density airlift bioreactor; Klein J et al.; In this article a hydrodynamic and rheological analysis of a continuous airlift bioreactor with high-cell-density system is presented . A highly flocculating recombinant strain of Sacharomyces cerevisiae containing genes for lactose transport (lactose permease) and hydrolysis (beta-galactosidase) was exploited to ferment lactose from cheese whey to ethanol . The magnetic particle-tracer method was used to assess the effect of operational conditions (air-flow rate, biomass concentration) on hydrodynamic behavior of an airlift bioreactor during the fermentation process . Measurements of liquid circulation velocity showed the existence of a critical value of biomass concentration at which a dramatic deceleration of net liquid flow appeared with increasing biomass quantity . Rheological analysis revealed exponential increase of viscosity of the yeast floc suspension at the same biomass concentration of about 73 g/dm(3) corresponding to 42.8% v/v of solid fraction . These facts have a particular importance for the successful processing of a high-cell-density airlift bioreactor as only a circulated flow regime will be favorable to keep the solid particles in suspension state and evenly distributed throughout the bioreactor . (c) 2004 Wiley Periodicals, Inc.

Zhonghua Yi Xue Za Zhi, 2004 Nov 2, 84(21), 1832 - 5
{In vitro immunocompatibility of a novel bioartificial liver reactor material: propylene-acidamide grafted polypropylene membrane.}; Peng CH et al.; OBJECTIVE: To evaluate the immunocompatibility of a novel bioartificial liver bioreactor material: propylene-acidamide grafted polypropylene membrane (PP-g-AAm) in vitro on peripheral blood mononuclear cells (PBMCs) . METHODS: Fifty milliliters of peripheral blood were collected from 30 healthy young people . PBMCs were extracted and inoculated on the 24-well culture plate preset with sterilized PP-g-AAm membrane and ungrafted polypropylene (PP) membrane . Automatic biochemical analyzer was used to detect the lactic dehydrogenase (LDH) value of the PMBCs after 2 and 6 hours . The PMBCs on these 2 kinds of membrane were cultured for 6 hours and then added with lipopolysaccharide and cultured continually . Six, twelve, and thirty-six hours later the supernatant was collected . ELISA was used to detect the values of tumor necrosis factor (TNF)alpha, interleukin (IL)-1beta, and IL-6 . Flow cytometry was used to detect the expression of CD69 antigen . Scanning electron microscopy was used to observe the adhesion of PMBCs on the materials . RESULTS: After 2 hours' epimembranous inoculation the LDH value was 43.50 U/L +/- 12.71 U/L in the PP group, significantly higher than that in the PP-g-AAm group (29.13 U/L +/- 8.74 U/L, P = 0.008) and the newly extracted PMBC group (0 h group, 19.89 U/L +/- 4.67 U/L, P = 0.000) . However, the difference between the 0 h group and PP-g-AAm group (P = 0.080) was insignificant . After 6 hours' epimembranous inoculation the LDH value was 50.25 U/L +/- 13.38 U/L in the PP group, and 32.50 U/L +/- 9.21 U/L in the PP-g-AAm group (P = 0.001), both significantly higher than that in the 0 h group (P = 0.000, and P = 0.019) . There was no significant difference between the values 2 hours and 6 hours after inoculation for the two groups . No expression of TNFalpha, IL-1beta, and IL-6 was found in the supernatant of the 2 groups without LPS stimulation . Expressions of TNFalpha, IL-1beta, and IL-6 could be found at a low level 12 hours after LPS stimulation for both groups and peaked 24 hours after LPS stimulation . The expressions of TNFalpha, IL-1beta, and IL-6 were lower in the PP-g-AAm group than in the PP group (P = 0.004, P = 0.003, and P = 0.022) . The TNFalpha, IL-1beta, and IL-6 levels all decreased after 36 hours . The CD69 antigen expression rates were 17.20% +/- 3.45%and 12.02% +/- 2.44% respectively in the PP group and PP-g-AAm group, both significantly higher than that of the blank control group (3.38% +/- 1.30%, both P = 0.000) . SEM showed that the PMBCs adhered on the PP-Aam membrane were significantly less then those adhered on the PP membrane . And the PMBCs adhered on the PP-g-AAm membrane were smaller and with less microvilli . CONCLUSION: PP-g-AAm membrane has weaker activation capability to PMBCs and has better immunocompatibility in comparison with the PP membrane.

IEEE Trans Nanobioscience, 2004 Dec, 3(4), 243 - 50
Complex permittivity measurement as a new noninvasive tool for monitoring in vitro tissue engineering and cell signature through the detection of cell proliferation, differentiation, and pretissue formation; Bagnaninchi PO et al.; In in vitro tissue engineering, microporous scaffolds are commonly used to promote cell proliferation and differentiation in three-dimensional structures . Classic measurement methods are particularly time consuming, difficult to handle, and destructive . In this study, a new nondestructive method based on complex permittivity measurement (CPM) is proposed to monitor and track the osteoblast and macrophage differentiation through their morphological variation upon cell attachment and proliferation inside the microporous scaffolds . CPM is performed using a vector network analyzer and a dielectric probe under sterile conditions in a laminar-flow hood . A suitable effective medium approximation (EMA) is applied to fit the data in order to extract the parameters of the different constituents . Our data show that the EMA depolarization factor can be monitored to assess the variation of cell morphology characterizing cell attachment . Discrimination between two batches of scaffolds seeded, respectively, with 2 million and 1 million osteoblast cells is possible; the ratio of their CPM-derived cell volume fractions is in agreement with the ratio of their cell seeding numbers . In addition, cell proliferation inside scaffolds seeded with osteoblasts cultured in alpha minimum essential medium and inside scaffolds seeded with osteoblasts cultured in alpha minimum essential medium supplemented to induce the formation of extracellular matrix is monitored via CPM over several days . CPM-determined cell volume fraction is compared to DNA assay cell counts . Extracellular matrix formation and cell presence was confirmed by scanning electron microscopy . A set of three signature parameters (epsilon'mem, epsilon'cyt, kappa'cyt) characteristic of cell line is extracted from CPM . Distinct signatures are recorded for osteoblasts and macrophages, thus confirming the ability of CPM to discriminate between different cell types . This study demonstrates the potential of CPM as a diagnostic tool to monitor quickly and noninvasively cell growth and differentiation inside microporous scaffolds . Our findings suggest that the use of CPM could be extended to many biomedical applications, such as drug detection and automation of tissue and bacterial cultures in bioreactors.

Appl Microbiol Biotechnol . 2005 Jan 4; {Epub ahead of print}
Enhancement of ginsenoside biosynthesis in high-density cultivation of Panax notoginseng cells by various strategies of methyl jasmonate elicitation; Wang W et al.; A single addition of 200 muM methyl jasmonate (MJA) to high-density cell cultures of Panax notoginseng enhanced ginsenoside production in both shake-flask (250 ml) and airlift bioreactor (ALR; 1 l working volume) . Repeated elicitation with two additions of 200 muM MJA during cultivation further induced the ginsenoside biosynthesis in both cultivation vessels . The content of ginsenosides Rg(1), Re, Rb(1) and Rd in the ALR was increased from, respectively, 0.18+/-0.01, 0.21+/-0.01, 0.21+/-0.02 and 0 mg per100 mg dry cell weight (DW) in untreated cell cultures (control) to 0.32+/-0.02, 0.36+/-0.02, 0.72+/-0.06 and 0.08+/-0.01 mg per100 mg DW with a single addition of MJA and further increased to 0.43+/-0.02, 0.46+/-0.03, 1.09+/-0.07 and 0.14+/-0.02 mg per100 mg DW with two additions of MJA . Interestingly, the activity of the Rb(1) biosynthetic enzyme (UDPG-ginsenoside Rd glucosyltransferase), was also increased with a single elicitation by MJA and increased again by a repeated elicitation, which coincided well with the trend in the increase in Rb(1) content . In order to further improve the cell density and ginsenoside production, a strategy of MJA repeated elicitation combined with sucrose feeding was adopted . The final cell density and total ginsenoside content in the ALR reached 27.3+/-1.5 g/l and 2.02+/-0.06 mg per100 mg DW; and the maximum production of ginsenoside Rg(1), Re, Rb(1) and Rd was 111.8+/-4.7, 117.2+/-4.6, 290.2+/-5.1 and 32.7+/-8.1 mg/l, respectively . The strategies demonstrated and the information obtained in this work are useful for the efficient large-scale production of bioactive ginsenosides by plant cell cultures.

Yi Chuan, 2004 Jan, 26(1), 75 - 83
{Analysis of Codon Usage in Potato and Its Application in the Modification of t-PA Gene.}; Bai X et al.; Bioperl-1.0 was used under Hongqi LINUX system to programm the codon analysis software.According to the analysis of 98 codon DNA sequences with this software,the codon usage in potato was calculated and 4 codons have been inferred to the optimal codons.The codons of tissue plasminogen activator (t-PA) gene sequence have been reconstructed according to the results.The t-PA gene sequence containing the optimal codons of potato will be used for t-PA production by potato bioreactor.

Biotechnol Bioeng . 2004 Dec 29; {Epub ahead of print}
Effect of culture pH on erythropoietin production by Chinese hamster ovary cells grown in suspension at 32.5 and 37.0 degrees C; Yoon SK et al.; To investigate the effect of culture pH in the range of 6.85-7.80 on cell growth and erythropoietin (EPO) production at 32.5 and 37.0 degrees C, serum-free suspension cultures of recombinant CHO cells (rCHO) were performed in a bioreactor with pH control . Lowering culture temperature from 37.0 to 32.5 degrees C suppressed cell growth, but cell viability remained high for a longer culture period . Regardless of culture temperature, the highest specific growth rate (mu) and maximum viable cell concentration were obtained at pH values of 7.00 and 7.20, respectively . Like mu, the specific consumption rates of glucose and glutamine decreased at 32.5 degrees C compared to 37.0 degrees C . In addition, they increased with increasing culture pH . Culture pH at 32.5 degrees C affected specific EPO productivity (q(EPO)) in a different fashion from that at 37 degrees C . At 37 degrees C, the q(EPO) was fairly constant in the pH range of 6.85-7.80, while at 32.5 degrees C, the q(EPO) was significantly influenced by culture pH . The highest q(EPO) was obtained at pH 7.00 and 32.5 degrees C, and its value was approximately 1.5-fold higher than that at pH 7.00 and 37.0 degrees C . The proportion of acidic EPO isoforms, which is a critical factor for high in vivo biological activity of EPO, was highest in the stationary phase of growth, regardless of culture temperature and pH . Although cell viability rapidly decreased in death phase at both 32.5 and 37.0 degrees C, the significant degradation of produced EPO, probably by the action of proteases released from lysed cells, was observed only at 37.0 degrees C . Taken together, through the optimization of culture temperature and pH, a 3-fold increase in maximum EPO concentration and a 1.4-fold increase in volumetric productivity were obtained at pH 7.00 and 32.5 degrees C when compared with those at 37.0 degrees C . These results demonstrate the importance of optimization of culture temperature and pH for enhancing EPO production in serum-free, suspension culture of rCHO cells . (c) 2004 Wiley Periodicals, Inc.

Biotechnol Bioeng . 2004 Dec 29; {Epub ahead of print}
Low-cost noninvasive optical CO(2) sensing system for fermentation and cell culture; Ge X et al.; High-throughput bioprocessing is a very promising technique for bioprocess development and optimization because of its high efficiency . The key to its development has been the availability of simple and inexpensive sensors to monitor the bioprocesses conducted in its small-scale bioreactors . Here we report on a low-cost noninvasive CO(2) sensing system suitable for any transparent vessel . The system was composed of a CO(2) sensing patch, a coaster, an interface, and a computer . The sensing film was prepared using the ion-pair technique . The coaster was a small self-made device with necessary optics and electronics for ratiometric measurements with a component cost of less than $100 . Results show that the system was stable and reliable despite its simplicity and low cost . The sensitivity of the CO(2) sensing system was not affected by pH, media type, or temperature . It was shown to be stable for at least 10 days, long enough for most bioprocesses . (c) 2004 Wiley Periodicals, Inc.

Huan Jing Ke Xue, 2004 Sep, 25(5), 102 - 5
{Printing and dyeing wastewater treatment using combined process of anaerobic bioreactor and MBR}; Zheng X et al.; This paper describes a labor-scale experiment for printing and dyeing wastewater treatment of woolen mill using a combined process of an anaerobic reactor and a membrane bioreactor (MBR) . The experimental results showed that when the concentration of COD, BOD5 and color in the influent were 128-321 mg/L, 36-95 mg/L and 40-70 dilution times (DT), the average concentrations of COD, BOD5, color and turbidity in the effluent were 36.9 mg/L, 3.7 mg/L, 21 DT and 0.24 NTU, respectively, and the corresponding removal rates were 80.3%, 95%, 59% and 99.3%, respectively . A new integrated membrane bioreactor by gravity drain of liquid level in the bioreactor was developed in this study . It not only lessens suction pump for effluent and controlling unit comparing to the traditional integrated membrane bioreactor, but also has the characters of high and continuous flux, concise configuration and simple operation and maintenance . According to the experimental results, the air flow rate through the membrane module is a significant factor to affect the flux rate and cake layer deposited on the membrane . With application of optimal air flow rate, it is effective to reduce membrane fouling and maintain high flux rate.

Biotechnol Bioeng . 2004 Dec 23; {Epub ahead of print}
Protein production and induction of the unfolded protein response in Trichoderma reesei strain Rut-C30 and its transformant expressing endoglucanase I with a hydrophobic tag; Collen A et al.; The effect of induction of protein production was studied in bioreactor cultures of T . reesei strain Rut-C30 and its transformant expressing endoglucanase I core domain (EGI, Cel7B) fused with a hydrophobic peptide tag . The tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation . The fungi were first grown on glucose-containing minimal medium after which rich medium with lactose as a carbon source was added to induce cellulase production . Production of extracellular protein and cellulase activity and the transcript levels of the major cellulase genes were analyzed during the cultivations . Induction of the cellulase genes followed a similar temporal pattern in both strains . The first phase of induction took place after addition of lactose as soon as glucose was depleted, and the second phase after lactose was consumed . Western analysis showed that a decreased amount of fusion protein was produced in the culture medium compared with the endogenous EGI, although the strain harbors several copies of the recombinant gene under the strong cbh1 promoter . The fusion protein appeared to accumulate within the cells, indicating impaired secretion of the protein . The mRNA levels of the UPR (unfolded protein response) target genes, bip1 and pdi1, and the level of the active form of hac1 transcript encoding the UPR transcription factor increased concurrently with induction of the cellulase genes in both strains, indicating increased requirement of the folding machinery under these conditions . However, only a minor increase in bip1 and pdi1 transcript level was observed in the transformant compared with the parental strain . (c) 2004 Wiley Periodicals, Inc.

J Agric Food Chem, 2004 Dec 29, 52(26), 7842 - 5
A novel angiotensin I converting enzyme inhibitory peptide from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate; Je JY et al.; Alaska pollack frame protein, which is normally discarded as an industrial byproduct in the processing of fish in plants, was hydrolyzed with pepsin . This was fractionated into five major types of Alaska pollack frame protein hydrolysates (APH-I, 10-30 kDa; APH-II, 5-10 kDa; APH-III, 3-5 kDa; APH-IV, 1-3 kDa; and APH-V, below 1 kDa) using an ultrafiltration membrane bioreactor system . Angiotensin I converting enzyme (ACE) inhibitory activities of the fractionated hydrolysates were investigated, and the fraction that exhibited the highest ACE inhibitory activity was further purified using consecutive chromatographic methods on SP-Sephadex C-25 column, Sephadex G-25 column, and high-performance liquid chromatography (HPLC) on an octadecylsilane column . Finally, we purified a novel ACE inhibitory peptide with an IC50 value of 14.7 microM, and the sequence of the peptide was Phe-Gly-Ala-Ser-Thr-Arg-Gly-Ala . In addition, the ACE inhibition pattern of the peptide was found to be noncompetitive.

Biotechnol Bioeng . 2004 Dec 17; {Epub ahead of print}
Culture of Escherichia coli under dissolved oxygen gradients simulated in a two-compartment scale-down system: Metabolic response and production of recombinant protein; Sandoval-Basurto EA et al.; A significant problem of large-scale cultures, but scarcely studied for recombinant E . coli, is the presence of gradients in dissolved oxygen tension (DOT) . In this study, the effect of DOT gradients on the metabolic response of E . coli and production of recombinant pre-proinsulin, accumulated as inclusion bodies, was determined . DOT gradients were simulated in a two-compartment scale-down system consisting of two interconnected stirred-tank bioreactors, one maintained at anoxic conditions and the other at a DOT of at least 6% . Cells were continuously circulated between both vessels to simulate circulation times (t(c)) of 20, 50, 90, and 180 sec . A complete kinetic and stoichiometric characterization was performed in the scale-down system as well as in control cultures maintained at constant DOT in the range of 0-20% . The performance of E . coli cultured under oscillating DOT was significantly affected, even at a t(c) of 20 sec corresponding to transient exposures of only 13.3 sec to anaerobic conditions . Specific growth rate decreased linearly with t(c) to a maximum reduction of 30% at the highest t(c) tested . The negative effect of DOT gradients was even more pronounced for the overall biomass yield on glucose and the maximum concentration and yield of pre-proinsulin . In these cases, the losses were 9%, 27%, and 20%, respectively, at t(c) of 20 sec and 65%, 94%, and 87%, respectively, at t(c) of 180 sec . Acetic, lactic, formic, and succinic acids accumulated during oscillatory DOT cultures, indicating that deviation of carbon flow to anaerobic metabolism was responsible for the observed losses . The results of this study indicate that even very short exposures to anaerobic conditions, typical of large-scale operations, can substantially reduce recombinant protein productivity . The information presented here is useful for establishing improved rational scale-up strategies and understanding the behavior of recombinant E . coli exposed to DOT gradients . (c) 2004 Wiley Periodicals, Inc.

J Ind Microbiol Biotechnol . 2004 Dec 17; {Epub ahead of print}
Cometabolic reduction of bromate by a mixed culture of microorganisms using hydrogen gas in a gas-lift reactor; Ginkel CG et al.; The discharge of bromate, a suspected carcinogen, will be restricted in the near future . To assess the possibility of biotechnological treatment of bromate-containing wastewaters, the removal of bromate by chlorate-reducing microorganisms was studied . The removal of bromate and chlorate was studied in laboratory gas-lift bioreactors supplied with hydrogen gas as electron donor in the absence of molecular oxygen . In these reactors, bromate was reduced cometabolically by chlorate-respiring microorganisms . To allow the cometabolic reduction of bromate, a chlorate:bromate molar ratio of at least 3:1 was required . The cometabolic conversion permitted almost complete reduction of bromate into bromide at hydraulic retention times of at least 6 h . Optimal bromate reduction activity was observed at approximately 35 degrees C . The pH optimum was between 7 and 8 . Bromate reduction in excess of 80% and a maximum bromate reduction rate of 2.3 g l(-1) day(-1) in a pilot-scale gas-lift bioreactor demonstrates that the process is sustainable.

Biotechnol Lett, 2004 Nov, 26(21), 1619 - 22
Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate; Kim YS et al.; Adventitious roots of ginseng were treated with methyl jasmonate (MJ) up to 150mu and cultured for 40days . Up to 100mu MJ inhibited the root growth but increase ginsenoside accumulation . In a two-stage bioreactor culture, total ginsenosides, after elicitation with 100 mu MJ peaked after 10days at 48mgg(-1) dry wt and then dropped sharply . Of the two groups of ginsenosides (Rb and Rg), higher amounts of Rb accumulated in the adventitious roots.

Altern Lab Anim, 2004 Mar, 32(1), 25 - 35
Evaluation of a flat membrane hepatocyte bioreactor for pharmacotoxicological applications: evidence that inhibition of spontaneously produced nitric oxide improves cell functionality; Canova N et al.; A laboratory-scale bioreactor was re-evaluated, with the aim of improving its use for the perfused culture of rat hepatocytes . In contrast to conventional culture systems, the flat membrane bioreactor (FMB) showed good functionality and biochemical competence during 2-3 days . Hepatocytes cultured in the FMB, specifically in a "sandwich" configuration, were functionally stable, as shown by a high rate of urea biosynthesis after challenge with NH4Cl, a low alanine-aminotransferase leakage and suppressed spontaneous nitric oxide (NO) production . Moreover, the time-course of the disappearance of cyclosporin A (CsA) from the perfusate demonstrated the high biotransformation capacity of cells in the FMB . The effect of CsA on the modulation of urea and spontaneous NO production demonstrated flexibility, in that minor changes could be observed at diverse time intervals and in a non-destructive way . The monitoring of nitrite levels during various steps of isolation and culture suggested that spontaneously produced NO has a negative impact on hepatocyte metabolic and functional integrity . In spite of the sophisticated techniques that are being used for the preparation of bioreactors, with hepatocytes surviving for longer periods, our data have shed light on some factors that could be important for the successful use of similar models for pharmacotoxicological and other biomedical applications.

J Neurosci Res, 2005 Jan 1-15, 79(1-2), 26 - 32
Culturing primary brain astrocytes under a fully controlled environment in a novel bioreactor; Sa Santos S et al.; We report the first approach for growth and maintenance of primary astrocytes on a fully controlled environment . For this purpose, cells were immobilized in Cytodex microcarriers and grown in a stirred tank bioreactor . The distribution of astrocytes at the microcarrier surface was visualized using confocal microscopy and glial fibrillary acidic protein (GFAP) labeling, a specific glial probe . Crucial bioreaction parameters such as agitation rate, microcarrier type, and concentration, as well as cell inoculum concentration were assessed . Cytodex 3 proved the best microcarrier for astrocyte growth, with the highest cell densities obtained for 6 g/l of Cytodex 3 using an inoculum of approx . 0.15 x 10(6) cells/ml in vessels operated at 60 rpm, using a refeed operational mode consisting of complete medium replacement every 5 days . Using such optimized conditions, cells were maintained in steady-state for approximately 24 days, allowing online monitoring and control of environmental variables such as temperature, pH, and O(2) . To test further the advantages of this fully controlled system, astrocytes were also subjected to hypoxic stress for 5 hr; the cell number was not affected by hypoxia but the glycolytic flux was enhanced during the stress imposed . The culture system described is a novel tool to study brain cell metabolism, allowing sampling over time and the monitoring of cellular behavior through stressful conditions and during recovery . (c) 2004 Wiley-Liss, Inc.

Hum Antibodies, 2004, 13(3), 69 - 79
Production of a human monoclonal IgM directed against human cardiac myosin in a hollow-fiber bioreactor for Membrane Anion Exchange Chromatography one-step purification; Jacobin MJ et al.; Purification of human IgM monoclonal antibodies (MAbs) has proved to be difficult . Since IgM Mabs tend to bind strongly to a variety of resin support surfaces, the number of chromatographic steps used in the purification of these biomolecules should be minimized . Here we describe procedures developed for the optimal production and purification of the human monoclonal IgM B7, which specifically binds to the myosin heavy chain of human ventricular myocardium . This property makes this antibody potentially useful for the diagnosis of myocardial necrosis . Several chromatographic techniques were evaluated (size exclusion, ion exchange, affinity chromatography) . The best results were obtained with anion exchange membrane chromatography using Sartobind Q15 (98% purity, 30% recovery) . IgM production was improved by the hollow fiber technology which permitted the use of serum-reduced medium and an increase in antibody concentration to an average production of 300-400 microg/ml, compared to 20 microg/ml in flask culture . Several flow-rates were also evaluated, the optimal being 20 ml/minute for 30% of recovery . Importantly, the purified IgM molecule was able to bind to human myosin in ELISA and Western-blotting, thus allowing the IgM to be kept intact for further radiolabeling.

Methods Mol Biol, 2005, 295, 55 - 70
Growing hybridomas; Entrican G et al.; Hybridomas can be grown in a number of ways to produce stocks of monoclonal antibodies . Smaller volumes may be produced in static flask culture, but if larger amounts are required, a bioreactor may be used.

Protein Expr Purif, 2005 Jan, 39(1), 61 - 70
Optimisation of insect cell growth in deep-well blocks: development of a high-throughput insect cell expression screen; Bahia D et al.; This report describes a method to culture insects cells in 24 deep-well blocks for the routine small-scale optimisation of baculovirus-mediated protein expression experiments . Miniaturisation of this process provides the necessary reduction in terms of resource allocation, reagents, and labour to allow extensive and rapid optimisation of expression conditions, with the concomitant reduction in lead-time before commencement of large-scale bioreactor experiments . This therefore greatly simplifies the optimisation process and allows the use of liquid handling robotics in much of the initial optimisation stages of the process, thereby greatly increasing the throughput of the laboratory . We present several examples of the use of deep-well block expression studies in the optimisation of therapeutically relevant protein targets . We also discuss how the enhanced throughput offered by this approach can be adapted to robotic handling systems and the implications this has on the capacity to conduct multi-parallel protein expression studies.

Toxicol Sci . 2004 Dec 8; {Epub ahead of print}
In Vitro Zonation and Toxicity in a Perfused Hepatocyte Co-Culture; Allen JW et al.; In vitro models that more closely represent the intricate architecture and functional diversity of the liver would be beneficial to toxicology . We have established a bioreactor culture system that recapitulates features of liver zonation in vitro, allowing investigation of compartmentalized drug metabolism and toxicity . In vitro zonation is induced by exposing hepatocyte cultures to oxygen and nutrient gradients in a parallel plate bioreactor . Gradients were modeled and experimentally validated over co-cultures of hepatocytes and fibroblasts that exhibit a stable hepatocyte phenotype in vitro . Co-cultures exposed to physiologic oxygen gradients exhibited zonal induction of CYP2B and CYP3A protein that mimics that found in vivo . Furthermore, exposure to a prototypic zonal hepatotoxin, APAP, resulted in maximal toxicity at the low-oxygen outlet region similar to centrilobular necrotic patterns observed in vivo . In conclusion, we have established a perfused hepatocyte co-culture system for molecular and applied investigation into zonation-dependent phenomena involving drug metabolism and toxicity.

Tissue Eng, 2004 Sep-Oct, 10(9-10), 1436 - 45
Tribology approach to the engineering and study of articular cartilage; Wimmer MA et al.; This study has been based on the assumption that articular motion is an important aspect of mechanotransduction in synovial joints . For this reason a new bioreactor concept, able to reproduce joint kinematics more closely, has been designed . The prototype consists of a rotating scaffold and/or cartilage pin, which is pressed onto an orthogonally rotating ball . By oscillating pin and ball in phase difference, elliptical displacement trajectories are generated that are similar to the motion paths occurring in vivo . Simultaneously, dynamic compression may be applied with a linear actuator, while two-step-motors generate the rotation of pin and ball . The whole apparatus is placed in an incubator . The control station is located outside . Preliminary investigations at the gene expression level demonstrated promising results . Compared with free-swelling control and/or simply compression-loaded samples, chondrocyte-seeded scaffolds as well as nasal cartilage explants exposed to interface motion both showed elevated levels of cartilage oligomeric matrix protein mRNA . The final design of the bioreactor will include four individual stations in line, which will facilitate the investigation of motion-initiated effects at the contacting surfaces in more detail.

Biomaterials, 2005 May, 26(15), 2509 - 16
An organic-inorganic hybrid scaffold for the culture of HepG2 cells in a bioreactor; Kataoka K et al.; Much interest has recently been shown in the potential utility of bioartificial liver (BAL) as a bridge support for patients and as a module for experimental purposes . A radial-flow bioreactor (RFB), one of the perfused bed/scaffold-type bioreactors, enables a highly functional three-dimensional culture as BAL . The functional capacity of bioreactors depends not only on their mechanistic structures but also on scaffolds packed in them . In the present study, we examined the possible utility of a new porous organic-inorganic-hybrid scaffold in an RFB . The scaffold was made from tetraethoxysilane (TEOS) and polydimethylsiloxane (PDMS) by a sol-gel method using sieved sucrose particles as a porogen . In the porous TEOS-PDMS hybrid scaffold, human hepatocellular carcinoma cells (HepG2) proliferated actively and formed cell clusters more efficiently than they did in a polyvinyl-alcohol scaffold . When cultivated in PDMS-TEOS, HepG2 cells secreted a approximately three-fold greater amount of albumin than that secreted in a monolayer culture . For potential application of BAL to pharmacological studies and future clinical use, it is essential to develop a method to propagate liver cells that maintain highly specific functions . The present results indicate that PDMS-TEOS may be a promising scaffold for developing such functional culture methods.

Biotechnol Bioeng, 2005 Jan 20, 89(2), 138 - 147
Development of a small-scale bioreactor: Application to in vivo NMR measurement; Gmati D et al.; A perfused bioreactor allowing in vivo NMR measurement was developed and validated for Eschscholtzia californica cells . The bioreactor was made of a 10-mm NMR tube . NMR measurement of the signal-to-noise ratio was optimized using a sedimented compact bed of cells that were retained in the bioreactor by a supporting filter . Liquid medium flow through the cell bed was characterized from a mass balance on oxygen and a dispersive hydrodynamic model . Cell bed oxygen demand for 4 h perfusion required a minimal medium flow rate of 0.8 mL/min . Residence time distribution assays at 0.8-2.6 mL/min suggest that the cells are subjected to a uniform nutrient environment along the cell bed . Cell integrity was maintained for all culture conditions since the release of intracellular esterases was not significant even after 4 h of perfusion . In vivo NMR was performed for (31)P NMR and the spectrum can be recorded after only 10 min of spectral accumulation (500 scans) with peaks identified as G-6P, F-6P, cytoplasmic Pi, vacuolar Pi, ATP(gamma) and ADP(beta), ATP(alpha) and ADP(alpha), NADP and NDPG, NDPG and ATP(beta) . Cell viability was shown to be maintained as (31)P chemical shifts were constant with time for all the identified nuclei, thus suggesting constant intracellular pH . (c) 2004 Wiley Periodicals, Inc.

Biotechnol Bioeng, 2005 Jan 20, 89(2), 157 - 63
Low-temperature pausing of cultivated mammalian cells; Hunt L et al.; There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37 degrees C and freezing in small batches . We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37 degrees C in a variety of cultivation vessels . High cell viability and exponential growth were observed after pausing a recombinant Chinese hamster ovary cell line (CHO-Clone 161) in a temperature range of 6-24 degrees C in microcentrifuge tubes for up to 3 weeks . After pausing in T-flasks at 4 degrees C for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37 degrees C . Adherent cultures of CHO-DG44 cells paused for 2 days at 4 degrees C in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4 degrees C or 24 degrees C in spinner flasks were efficiently transfected by the calcium phosphate-DNA coprecipitation method, yielding reporter protein levels comparable to those from nonpaused cultures . Finally, cultures of a recombinant CHO cell line (CHO-YIgG3) paused for 3 days at 4 degrees C, 12 degrees C, or 24 degrees C in bioreactors achieved the same cell mass and recombinant protein productivity levels as nonpaused cultures . The success of this approach to cell storage with rodent and human cell lines points to a general biological phenomenon which may have a wide range of applications for cultivated mammalian cells . (c) 2004 Wiley Periodicals, Inc.

J Hepatol, 2004 Dec, 41(6), 950 - 6
Development of a scaled up liver device incorporating cryo-preserved pig liver micro-organs; Gershonowitz A et al.; BACKGROUND/AIMS: Currently there is no effective therapy for most patients with fulminant or end stage liver disease . METHODS: Pig liver micro-organs (LMOs), which preserve liver micro-architecture and ensure a maximal 150-200mum distance from a source of nutrients and gases have been prepared and a method to cryo-preserve them has been developed . A new scaled-up extra-corporeal liver device termed aLIVE-H in which LMOs are exposed to liver-like hemodynamic conditions has also been developed . The purpose of this work is to test the safety and function of cryo-preserved LMOs and how the hemodynamic properties of the scaled up aLIVE device affect their function . RESULTS: Pig LMOs in aLIVE-H, transcribe albumin and Factor V at similar levels, irrespective of their position within the bioreactor, indicating that the hemodynamic features of the aLIVE-H device allow for homogeneous plasma distribution and proper function at different locations . Cryo-preserved LMOs transcribe albumin and Factor V at levels comparable to those transcribed by a normal pig liver . Connecting the aLIVE-H bioreactor to normal pigs did not affect key blood components and biochemical parameters . CONCLUSIONS: An extra-corporeal liver device aLIVE-H which imitates the hemodynamic and functional properties of the normal liver and incorporates cryo-preserved LMOs has been developed and characterized . aLIVE-H was found to perform key synthetic liver functions.

Chemosphere, 2005 Jan, 58(3), 299 - 310
Carbon/electron source dependence of polychlorinated biphenyl dechlorination pathways for anaerobic granules; Nollet H et al.; The effect of acclimating anaerobic granules from commercial bioreactors with different carbon/electron sources on their ability to reductively dechlorinate a tri-(2,3,4-CB) and heptachlorobiphenyl (2,2',3,3',4,5,6-CB) was studied . The anaerobic granules were first grown in upflow anaerobic sludge blanket (UASB) reactors fed with two different mixtures of carbon/electron sources, i.e., propionate/butyrate/methanol and formate/methanol . Differences in dechlorination patterns for 2,2',3,3',4,5,6-CB were observed in batch experiments inoculated with granules from these two sets of UASB reactors . Variation of the carbon/electron source, during the dechlorination process, had no effect on the dechlorination pathway, but the extents and rates of dechlorination were highest for ethanol and formate and lowest for pyruvate fed batches . Pre-acclimation of different anaerobic sludges to polychlorinated biphenyls (PCBs) shortened the lag period, but did not influence the PCB dechlorination pathway . This is the first time that similar acclimation conditions for several anaerobic microbial communities prior to inoculation were reported to yield similar substrate specificities for the reductive dechlorination of specific PCB congeners . This research demonstrates a successful strategy for the development of biocatalysts to serve as the inoculum of partially decontaminated sites in order to provide microorganisms with specificities complementary to those of naturally occurring dechlorinators.

Water Sci Technol, 2004, 50(9), 17 - 23
Use of microwave pretreatment for enhanced anaerobiosis of secondary sludge; Park B et al.; This work elucidates the effects of pretreatment of secondary sludge by microwave irradiation on anaerobic digestion . The soluble chemical oxygen demand (COD) concentration increased up to 22% as microwave irradiation time increased, which indicated the sludge particles disintegrated . Three identical automated bioreactors with working volume of 5 l were used as anaerobic digesters at mesophilic temperature (35 degrees C) . The reactors were separately fed with sludge with microwave pretreated- and control- sludge at different hydraulic retention times (HRT) . The volatile solid (VS) reduction in the control operation was approximately 23.2 +/- 1.3%, while it was 25.7 +/- 0.8% for the reactors with the pretreated sludge . The average biogas production rate with the pretreated sludge at 8, 10, 12, and 15 days HRTs was 240 +/- 11, 183 +/- 9, 147 +/- 8, and 117 +/- 7 ml/l/d respectively, while those with the control sludge were 134 +/- 12 and 94 +/- 7 ml/l/d at 10 and 15 days HRTs . Maximum rates of COD removal and methane production with the pretreated sludge were 64% and 79% higher than those of the control system, respectively.

Appl Microbiol Biotechnol . 2004 Dec 2; {Epub ahead of print}
Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of Panax ginseng in 5-l balloon type bubble bioreactors; Thanh NT et al.; The effects of methyl jasmonate (MJ) elicitation on the cell growth and accumulation of ginsenoside in 5-l bioreactor suspension cultures of Panax ginseng were investigated . Ginsenoside accumulation was enhanced by elicitation by MJ (in the range 50-400 muM); however, fresh weight, dry weight and growth ratio of the cells was strongly inhibited by increasing MJ concentration . The highest ginsenoside yield was obtained at 200 muM MJ . In the second experiment, 200 muM MJ was added on day 15 during the cultivation . The ginsenoside, Rb group, and Rg group ginsenoside content increased 2.9, 3.7, and 1.6 times, respectively, after 8 days of MJ treatment . Rb group gisnsenosides accumulated more than Rg group ginsenosides . Among Rb group ginsenosides, Rb1 content increased significantly by four times but the contents of Rb2, Rc and Rd increased only slightly . Among Rg group ginsenosides, Rg1 and Re showed 2.3-fold and 3.0-fold increments, respectively, whereas there was only a slight increment in Rf group ginsenosides . These results suggest that MJ elicitation is beneficial for ginsenoside production using 5-l bioreactor cell suspension cultures.

Biomaterials, 2005 May, 26(14), 2001 - 12
Low-density cultures of bovine chondrocytes: effects of scaffold material and culture system; Hu JC et al.; Chondrocytes were seeded on either agarose or polyglycolic acid (PGA) unwoven meshes at 10 million cells/ml of scaffold volume to evaluate the effect that these two biomaterials have on the low-density culture of chondrocytes in a rotating-wall bioreactor . For both static and bioreactor culture, agarose constructs contained more glycosaminoglycan than their PGA counterparts . However, the PGA constructs contained more collagen for both culture conditions when compared to agarose . For the low seeding density of this study, PGA constructs cultured in the bioreactor did not outperform static cultures when comparing collagen content after 8 weeks . The mechanical properties of the PGA constructs also did not improve with culture time . Similar results were observed with the agarose culture, though both static- and bioreactor-culture agarose constructs exhibited increases in aggregate modulus at the end of the culture period . As in PGA culture, chondrocytes cultured in agarose may require a higher density to reap the benefits of the bioreactor environment.

Biomaterials, 2005 May, 26(14), 1857 - 75
The roles of tissue engineering and vascularisation in the development of micro-vascular networks: a review; Kannan RY et al.; The construction of tissue-engineered devices for medical applications is now possible in vitro using cell culture and bioreactors . Although methods of incorporating them back into the host are available, current constructs depend purely on diffusion which limits their potential . The absence of a vascular network capable of distributing oxygen and other nutrients within the tissue-engineered device is a major limiting factor in creating vascularised artificial tissues . Though bio-hybrid prostheses such as vascular bypass grafts and skin substitutes have already been developed and are being used clinically, the absence of a capillary bed linking the two systems remains the missing link . In this review, the different approaches currently being or that have been applied to vascularise tissues are identified and discussed.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1888 - 92
Temperature effects on product-quality-related enzymes in batch CHO cell cultures producing recombinant tPA; Clark KJ et al.; Culture conditions that affect product quality are important to the successful operation and optimization of bioreactors . Previous studies have demonstrated that enzymes, such as proteases and sialidases, accumulate in batch bioreactors . These enzymes are known to be detrimental to the quality of recombinant glycoproteins . Bioreactor temperature has been used to control cell growth and recombinant protein production rates . However, the effect of culture temperature on the production of proteases and sialidases has not been investigated . In this study, Chinese hamster ovary cells were cultured with a temperature profile that decreased from 37 to 34 degrees C over 8 days and with a constant temperature of 37 degrees C . Analysis of extracellular protease activity indicated that two major proteases were present (50 and 69 kDa) . The 50 kDa protease activity decreased similarly with time for both culture conditions . The 69 kDa protease activity increased with time for both culture conditions . The constant-temperature cultures had significantly lower 69 kDa protease levels compared to the ramped-temperature cultures in the early stationary phase . Intracellular sialidase activity was present in both cultures . The intracellular sialidase activity increased dramatically for both culture conditions immediately after the cells were inoculated into fresh medium . The initial peak in intracellular sialidase activity was followed by a first-order decay . The intracellular sialidase activities for the two culture conditions were not significantly different . The production of recombinant tissue type plasminogen activator was not significantly different for the two culture conditions. , , . Thus, the previously hypothesized advantages that lower culture temperatures have reduced protease activity and improved productivity do not appear to be universal.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1802 - 9
A novel rotating-shaft bioreactor for two-phase cultivation of tissue-engineered cartilage; Chen HC et al.; A novel rotating-shaft bioreactor (RSB) was developed for two-phase cultivation of tissue-engineered cartilage . The reactor consisted of a rotating shaft on which the chondrocyte/scaffold constructs (7.5 mm diameter x 3.5 mm thickness) were fixed and a reactor vessel half-filled with medium . The horizontal rotation of the shaft resulted in alternating exposure of the constructs to gas and liquid phases, thus leading to efficient oxygen and nutrient transfer, as well as periodically changing, mild shear stress exerting on the construct surfaces (0-0.32 dyn/cm2 at 10 rpm), as revealed by computer simulation . Strategic operation of the RSB (maintaining rotating speed at 10 rpm for 3 weeks and lowering the speed to 2 rpm in week 4) in combination with higher seeding density (6 x 10(6) chondrocytes/scaffold) and medium perfusion resulted in uniform cell distribution and increased glycosaminoglycan (3.1 mg/scaffold) and collagen (7.0 mg/scaffold) deposition . The 4-week constructs resembled native cartilages in terms of not only gross appearance and cell morphology but also distributions of glycosaminoglycan, total collagen, and type II collagen, confirming the maintenance of chondrocyte phenotype and formation of cartilage-like constructs in the RSB cultures . In summary, the novel RSB may be implicated for in vitro study of chondrogenesis and de novo cartilage development under periodic mechanical loading . With proper optimization of the culture conditions, a RSB may be employed for the production of cartilage-like constructs.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1788 - 96
High-level scu-PA production by butyrate-treated serum-free culture of recombinant CHO cell line; Kim JS et al.; The MGpUK-5 cell line, transformed with a single-chain urokinase-type plasminogen activator (scu-PA) minigene, generated mRNA transcripts and scu-PA titers corresponding to 65% or 86% of the amount generated before serum-free adaptation, despite significant loss of scu-PA gene copies during adaptation to serum-free culture . To further augment scu-PA production, a culture strategy employing sodium butyrate was explored . In 60-mL spinner flask cultures, sodium butyrate in the concentration range 1-10 mM allowed scu-PA production 2- to 3-fold higher than that in the negative control culture . Its productivity-enhancing activity was dependent on cell density in a range of 1-5 x 10(6) cells/mL, generating 72,200 +/- 8,100 IU/mL (480 +/- 50 mg/L) in 60-mL spinner flask cultures . To confirm this result, cells were grown to 4.4 x 10(6) cells/mL and treated with 5 mM sodium butyrate in a 2.5-L perfusion culture . The scu-PA titer increased more than 2-fold, and specific production rate of scu-PA increased 3-fold by this treatment . Overall, this perfusion culture gave rise to 1.7 x 10(8) IU scu-PA (1.1 g), comparable to total scu-PA production in a batch butyrate-treated culture performed at a 25-L bioreactor scale (1.3-3.5 g) . Our results suggest that sodium butyrate treatment on high-density culture enables scu-PA production in gram quantities.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1725 - 32
Polymer development for enhanced delivery of phenol in a solid-liquid two-phase partitioning bioreactor; Prpich GP et al.; Two-Phase Partitioning Bioreactors (TPPBs) have traditionally been used to partition toxic concentrations of xenobiotics from a cell-containing aqueous phase by means of an immiscible organic solvent and to deliver these substrates back to the cells on the basis of metabolic demand and the maintenance of thermodynamic equilibrium between the phases . A limitation of TPPBs, which use organic liquid solvents, is the possibility that the solvent can be bioavailable, and this has therefore limited organic liquid TPPBs to the use of pure strains of microbes . Solid polymer beads have recently been introduced as a replacement for liquid organic solvents, offering similar absorption properties but with the capability to be used with mixed microbial populations . The present work was aimed at identifying a polymer with a greater capacity for and more rapid uptake and release of phenol for use as the second phase in a mixed culture TPPB . Polarity and hydrogen bonding capabilities between polymer and phenol were considered in the screening and selection process of candidate polymers . Hytrel (a copolymer of poly(butylene terephthalate) and butylene ether glycol terephthalate) polymer beads, offered improved capacity (19 mg phenol/g polymer at a fixed initial phenol concentration of 2000 mg/L) and a greater diffusivity (1.54 x 10(-7) cm2/s) when compared to the capacity and diffusivity of previously used EVA (ethylene vinyl acetate) beads (12.4 mg phenol/g polymer and 3.73 x 10(-9) cm2/s, respectively) . Hytrel polymer beads were then used in a TPPB for the investigation of various substrate feeding strategies (fed-batch, bead replacement, and concentrated spikes of phenol), with rapid and complete phenol degradation shown in all cases.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1718 - 24
A novel parallel shaken bioreactor system for continuous operation; Akgun A et al.; A novel continuous bioreactor system was developed as a shaken culture vessel for the investigation of the growth kinetics and product formation of microorganisms in milliscale . The novel bioreactor system mainly consists of a specially designed 250-mL shake flask with two inlets, one for gas supply and one for medium supply, and one combined outlet on the side of flask for exhaust gas and culture liquid . As a result of the circulating motion of the fermentation broth in the shake flask, the maximum liquid height reaches the edge of the outlet and the fermentation broth is accelerated into the outlet by centrifugal force . Additionally, the excess fermentation broth leaving the culture vessel is continuously driven by the exhaust gas . Because of the small scale and the simple handling it is possible to operate many of these shaken bioreactor vessels simultaneously . By using parallel vessels operated at different dilution rates on the same shaker, the data for a complete biomass over dilution rate (X-D) diagram of a biological culture can be evaluated in an efficient manner, thus saving money, materials, and time . Continuous fermentations of the yeast Saccharomyces cerevisiae H1022 (ATCC 32167) in the shaken bioreactor system and in a conventional stirred tank fermentor showed very similar results.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1710 - 7
Experimental study of a ceramic microsparging aeration system in a pilot-scale animal cell culture; Nehring D et al.; The oxygen supply of cell cultures with the aid of free gas bubbles is an efficient process strategy in pharmaceutical production . If the cell-damaging impact of gas bubbles is reduced, direct aeration becomes a practical solution with scale-up potential and comparatively high oxygen transfer rates . In this paper a microsparging aeration system made of porous ceramic was compared with bubble-free membrane aeration . The sparging system was used for the long-term cultivation of mammalian cells in 2- to 100-L scale bioreactors and produced bubble sizes of 100-500 microm in diameter . Using a scale of 2.5 and 30 L, a cell density of 2.6 x 10(6) cells/mL was attained . When a 100-L scale was used, a density of 1.1 x 10(6) cells/mL was achieved, whereas a comparable membrane-aerated system showed a cell density of 2.2 x 10(6) cells/mL . At relatively low agitation rates of less than 70 rpm in the sparged bioreactors, a homogeneous and constant oxygen concentration was kept in the medium . As a result of the different foam-forming tendency caused by the lower gas flow of the ceramic sparger compared to that of the standard aeration systems, we were able to develop an appropriate process control strategy . Furthermore, oxygen transfer measurements for the common stainless steel sparger and the ceramic sparger showed a 3-fold higher oxygen transfer coefficient for the ceramic sparger.

Aviakosm Ekolog Med, 2000, 34(5), 51 - 9
{Transmission and exchange of genetic information during bacterial conjugation in ground-based simulations of the factors of orbital flight}; Zerov IuP et al.; Control laboratory experiments on bacterial conjugation under simulated spaceflight conditions were performed with the use of new equipment (bioreactor RECOMB-2 and container BIOMAGNISTAT) within the RSA-NASA science program . External parameters were selected and the plan of simulation of a space experiment was verified to ensure high efficiency of the conjugative transfer of chromosomal and plasmid DNA and storage of hybrids on the ground . Genetic analysis of conjugative hybrids E . coli supported the hypothesized possibility of transfer of a whole bacterial chromosome during conjugation that will lead to relative stabilization of the diploid state . Earlier this hypothesis was used to interpret results of experiments performed on MIR in 1992-1993 . Hence, the ground laboratory investigations proved the conclusion about high probability of transfer of large fragments or even a whole chromosome during space flight . Screening of the geomagnetic field by BIOMAGNISTAT increases the probability of conjugative contacts between cells and is likely to slightly inhibit the processes of recombination.

ASAIO J, 2002 Jan-Feb, 48(1), 12 - 6
Application of stereolithography for scaffold fabrication for tissue engineered heart valves; Sodian R et al.; A crucial factor in tissue engineering of heart valves is the functional and physiologic scaffold design . In our current experiment, we describe a new fabrication technique for heart valve scaffolds, derived from x-ray computed tomography data linked to the rapid prototyping technique of stereolithography . To recreate the complex anatomic structure of a human pulmonary and aortic homograft, we have used stereolithographic models derived from x-ray computed tomography and specific software (CP, Aachen, Germany) . These stereolithographic models were used to generate biocompatible and biodegradable heart valve scaffolds by a thermal processing technique . The scaffold forming polymer was a thermoplastic elastomer, a poly-4-hydroxybutyrate (P4HB) and a polyhydroxyoctanoate (PHOH) (Tepha, Inc., Cambridge, MA) . We fabricated one human aortic root scaffold and one pulmonary heart valve scaffold . Analysis of the heart valve included functional testing in a pulsatile bioreactor under subphysiological and supraphysiological flow and pressure conditions . Using stereolithography, we were able to fabricate plastic models with accurate anatomy of a human valvular homograft . Moreover, we fabricated heart valve scaffolds with a physiologic valve design, which included the sinus of Valsalva, and that resembled our reconstructed aortic root and pulmonary valve . One advantage of P4HB and PHOH was the ability to mold a complete trileaflet heart valve scaffold from a stereolithographic model without the need for suturing . The heart valves were tested in a pulsatile bioreactor, and it was noted that the leaflets opened and closed synchronously under subphysiological and supraphysiological flow conditions . Our preliminary results suggest that the reproduction of complex anatomic structures by rapid prototyping techniques may be useful to fabricate custom made polymeric scaffolds for the tissue engineering of heart valves.

Space Med Med Eng (Beijing), 2001 Apr, 14(2), 149 - 53
{Prospect of the Advanced Life Support Program Breadboard Project at Kennedy Space Center in USA}; Guo SS et al.; The Breadboard Project at Kennedy Space Center in NASA of USA was focused on the development of the bioregenerative life support components, crop plants for water, air, and food production and bioreactors for recycling of wastes . The keystone of the Breadboard Project was the Biomass Production Chamber (BPC), which was supported by 15 environmentally controlled chambers and several laboratory facilities holding a total area of 2150 m2 . In supporting the Advanced Life Support Program (ALS Program), the Project utilizes these facilities for large-scale testing of components and development of required technologies for human-rated test-beds at Johnson Space Center in NASA, in order to enable a Lunar and a Mars mission finally.

Cell Motil Cytoskeleton, 2001 Nov, 50(3), 161 - 72
Saturable binding of the echinoderm microtubule-associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments; Eichenmuller B et al.; The echinoderm microtubule-associated protein (EMAP) is a 75-kDa, WD-repeat protein associated with the mitotic spindle apparatus . To understand EMAP's biological role, it is important to determine its affinity for microtubules (MTs) and other cytoskeletal components . To accomplish this goal, we utilized a low-cost, bubble-column bioreactor to express EMAP as a hexahistidine fusion (6his) protein in baculovirus-infected insect cells . After optimizing cell growth conditions, up to 30 mg of EMAP was obtained in the soluble cell lysate from a 1-liter culture . EMAP was purified to homogeneity in a two-step process that included immobilized metal-affinity chromatography (IMAC) and anion-exchange chromatography . In vitro binding studies on cytoskeletal components were performed with the 6his-EMAP . EMAP bound to MTs, but not actin or vimentin filaments, with an intrinsic dissociation constant of 0.18 microM and binding stoichiometry of 0.7 mol EMAP per mol tubulin heterodimer . In addition, we show that a strong MT binding domain resides in the 137 amino acid, NH(2)-terminus of EMAP and a weaker binding site in the WD-domain . Previous work has shown that the EMAP concentration in the sea urchin egg is over 4 microM . Together, these results show that there is sufficient EMAP in the egg to regulate the assembly of a large pool of maternally stored tubulin .

Biotechnol Bioeng, 2002 Mar 20, 77(6), 704 - 16
Inhibiting apoptosis in mammalian cell culture using the caspase inhibitor XIAP and deletion mutants; Sauerwald TM et al.; Lower yields and poorer quality of biopharmaceutical products result from cell death in bioreactors . Such cell death may occur from necrosis but is more commonly associated with apoptosis . During the process of programmed cell death or apoptosis, caspases become activated and cause a cascade of events that eventually destroy the cell . XIAP is the most potent caspase inhibitor encoded in the mammalian genome . The effectiveness of XIAP and its deletion mutants was examined in two cell lines commonly utilized in commercial bioreactors: Chinese hamster ovary (CHO) and 293 human embryonic kidney (293 HEK) cells . CHO cells undergo apoptosis as a result of various insults, including Sindbis virus infection and serum deprivation . In this study, we demonstrate that 293 HEK cells undergo apoptosis during Sindbis virus infection and exposure to the toxins, etoposide and cisplatin . Two deletion mutants of XIAP were created; one containing three tandem baculovirus iap repeat (BIR) domains and the other containing only the C-terminal RING domain, lacking the BIRs . Viability studies were performed for cells expressing each mutant and the wild-type protein on transiently transfected cells, as stable pools, or as stable clonal cell populations after induction of apoptosis by serum deprivation, Sindbis virus infection, etoposide, and cisplatin treatment . Expression of the wild-type XIAP inhibited apoptosis significantly; however, the XIAP mutant containing the three BIRs provided equivalent or improved levels of apoptosis inhibition in all cases . Expression of the RING domain offered no protection and was pro-apoptotic in transient expression experiments . With the aid of an N-terminal YFP fusion to each protein, distribution within the cell was visualized, and the wild-type and mutants showed differing intracellular accumulation patterns . While the wild-type XIAP protein accumulated primarily in aggregates in the cytosol, the RING mutant was enriched in the nucleus . In contrast, the deletion mutant containing the three BIRs was distributed evenly throughout the cytosol . Thus, protein engineering of the XIAP protein can be used to alter the intracellular distribution pattern and improve the ability of this caspase inhibitor to protect against apoptosis for two mammalian cell lines .

Biotechnol Bioeng, 2002 Mar 20, 77(6), 685 - 92
Effect of genetic background and culture conditions on the production of herpesvirus-based gene therapy vectors; Ozuer A et al.; Herpes simplex virus type-1 (HSV-1) represents a unique vehicle for the introduction of foreign DNA to cells of a variety of tissues . The nature of the vector, the cell line used for propagation of the vector, and the culture conditions will significantly impact vector yield . An ideal vector should be deficient in genes essential for replication as well as those that contribute to its cytotoxicity . Advances in the engineering of replication-defective HSV-1 vectors to reduce vector-associated cytotoxicity and attain sustained expression of target genes make HSV-1 an attractive gene-delivery vehicle . However, the yield of the less-cytotoxic vectors produced in standard tissue-culture systems is at least three order of magnitudes lower than that achieved with wild-type virus . The low overall yield and the complexity involved in the preparation of HSV vectors at high concentrations represent major obstacles in use of replication-defective HSV-derived vectors in gene therapy applications . In this work, the dependence of the vector yield on the genetic background of the virus is examined . In addition, we investigated the production of the least toxic, lowest-yield vector in a CellCube bioreactor system . After initial optimization of the operational parameters of the cellcube system, we were able to attain virus yields similar to or better than those values attained using the tissue culture flask system for vector production with significant savings with respect to time, labor, and cost .

Environ Toxicol Chem, 2002 Jan, 21(1), 1 - 8
Alpha,beta-unsaturated sulfophenylcarboxylates as degradation intermediates of linear alkylbenzenesulfonates: evidence for omega-oxygenation followed by beta-oxidations by liquid chromatography-mass spectrometry; Eichhorn P et al.; Liquid chromatography with an electrospray interface to a mass spectrometer (LC-ES-MS) and LC-ES coupled to a tandem MS (LC-ES-MS/MS) were used to detect and identify intermediates excreted transiently during the aerobic degradation of linear alkylbenzenesulfonates (LAS) in fixed-bed bioreactors (FBBR) . The inoculum for the FBBR was the microflora of the River Rhine, Germany . Two major phenomena were observed on the addition of 100 mg/L LAS to the system, sorption and then biodegradation . Disappearance due to sorption was followed in an inhibited FBBR . Biodegradation of LAS started on day 7 and was accompanied by the transient excretion of intermediates, which were later largely degraded . We detected not only the sulfophenylcarboxylates (SPCs) observed previously but also the alpha,beta-unsaturated SPCs (SPC-2H), which have not been reported before . Experiments with the (4-sulfophenyl)dodecanes (C12-LAS), which had minor contaminants of C11-LAS, showed C12-, C10-, C8-, C6-, and C4-SPCs when LAS was degraded as well as traces of C9-, C7-, and C5-SPCs . Signals from the SPC-2H species were usually some 10% of those from the corresponding SPCs . Samples from these experiments were also examined by gas chromatography-mass spectrometry (GC-MS), but no desulfonated intermediates were detected . We interpret the data to mean that the only attack on LAS was by (omega-oxygenation; there was no visible initial desulfonation . The products of omega-oxygenation were oxidized to the corresponding SPC and subject to beta-oxidation, as evidenced not only by the pattern of C-2 units in the excreted SPCs but also in the corresponding series of SPC-2H, representing the intermediates in beta-oxidation.

Crit Rev Biotechnol, 2001, 21(4), 233 - 94
Hydrodynamic and mass transfer characteristics of three-phase gaslift bioreactor systems; Petersen EE et al.; This review focuses on the hydrodynamic and mass transfer characteristics of various three-phase, gaslift fluidized bioreactors . The factors affecting the mixing and volumetric mass transfer coefficient (k(L)a), such as liquid properties, solid particle properties, liquid circulation velocity, superficial gas velocity, bioreactor geometry, are reviewed and discussed . Measurement methods, modeling and empirical correlations are reviewed and compared . To the authors' knowledge, there is no 'generalized' correlation to calculate the volumetric mass transfer coefficient, instead, only 'type-specific' correlations are available in the literature . This is due to the difficulty in modeling the gaslift bioreactor, caused by the variation in geometry, fluid dynamics, and phase interactions . The most important design parameters reported in the literature are: gas hold-up, liquid circulation velocity, 'true' superficial gas velocity, mixing, shear rate, aeration rate and volumetric mass transfer coefficient, k(L)a.

Int J Artif Organs, 2001 Nov, 24(11), 807 - 13
The effect of serum from liver cancer patients on the growth and function of primary and immortalised hepatocytes; Grant MH et al.; A limiting factor in the efficacy of bioartificial liver (BAL) for the treatment of liver failure is the toxicity of the patients' serum to the hepatocytes in the device . This study investigates the interaction of liver cancer patient serum with primary and immortalised rat hepatocytes . Liver cancer serum increased the growth rate of immortalised hepatocytes, without affecting reduced glutathione levels . The activities of DT-diaphorase and pi glutathione-S-transferase (GST), enzymes associated with de-differentiation, were also increased . Exposure of primary hepatocytes to liver cancer serum resulted in a decrease in cytochrome P450 (CYP) content, and in P450 dependent metabolism of testosterone . Formation of 2-alpha- and 6-beta- hydroxy testosterone was decreased . These reactions are predominantly associated with CYP 2C11 and 3A1 respectively in normal rat liver . The activity of total GST was also decreased, although that of the pi isoenzyme of GST was not affected . Our results suggest that exposure of hepatocytes in a bioreactor to liver cancer patient serum will result in overgrowth of cells, if proliferating cells are being used, and in de-differentiation . The serum may have to be pretreated with adsorbants to remove toxins prior to BAL treatment.

Int J Artif Organs, 2001 Nov, 24(11), 793 - 8
Experimental evaluation of a cell module for hybrid liver support; Gerlach JC et al.; Aim of the study was to evaluate a hybrid liver support system in a porcine model of acute liver failure, after hepatectomy . Pigs with a body weight of 70+/-18 kg underwent total hepatectomy and porto-cavo-caval shunting as well as ligation of the bile duct and the hepatic artery . Control animals were connected to the system (including capillary membrane plasma separation) containing a four compartment bioreactor with integral oxygenation and decentralized mass exchange but without liver cells . The treatment group received hybrid liver support with the same system including 370+/-42 g primary isolated porcine parenchymal liver cells in co-culture with hepatocyte nursing cells, tissue engineered to liver- like structures at high density . Treatment started after complete recovery from anesthesia and was performed continuously . A positive influence on peripheral vascular resistance and a reduced need of catecholamine dosage was observed in the treatment group . Hybrid liver support with a cell module upscaled for clinical application significantly prolonged survival time in animals after hepatectomy with the longest survival being 26 hours in the control group an 57 hours in the treatment group.

Ann N Y Acad Sci, 2001 Nov, 944, 334 - 43
Effect of flow configuration and membrane characteristics on membrane fouling in a novel multicoaxial hollow-fiber bioartificial liver; MacDonald JM et al.; A novel "multicoaxial hollow fiber bioreactor" has been developed consisting of four concentric tubes, the two innermost tubes are called hollow fibers . Bioartificial livers are created by culturing liver progenitors in the space between the two innermost hollow fibers and with culture media contained in the two compartments (intracapillary and extracapillary) sandwiching the cell compartment . The outermost compartment is used for gas exchange . A hydrodynamic model has recently been established to predict the optimum hydraulic permeability and bioreactor operational parameters to create the physicochemical environment found in the liver acinus . However, perfusion with serum-free hormonally-defined media and inoculation of cells introduces membrane fouling into the equation, and this parameter must be incorporated into the model . Using commercially available semipermeable hollow fibers (1 mm {0.65 microm pores} and 3 mm {0.1 microm pores} outer diameters {o.d}), the primary cause of resistance is the middle hollow fiber . Preliminary studies using bioreactors inoculated with isolated rat hepatocytes and perfused with serum-containing culture media demonstrated that the middle hollow fiber is the primary site of fouling, and this fouling ultimately causes cell mortality by blocking the transfer of nutrients . Experiments were performed to determine the best commercially available middle hollow fiber for construction of bioreactors and two 3-mm outer-diameter middle hollow fibers were compared: polypropylene and polysulfone, with 0.2 microm and 0.1 microm pore sizes, respectively . Dead-ended and cross flow configurations were compared for their effectiveness at reducing membrane fouling in the middle hollow fiber by determining the change in resistance with time . The results demonstrate that the 0.2-microm pore size polypropylene hollow fiber is the best choice for construction of the multicoaxial hollow-fiber bioreactor, and that cross flow results in two orders of magnitude lower resistance than dead-ended flow after 36 h.

Ann N Y Acad Sci, 2001 Nov, 944, 308 - 19
Development of a hybrid liver support system; Sauer IM et al.; Hybrid liver systems are being developed as temporary extracorporeal liver support therapy . The overview given here emphasizes the development of both hepatocyte culture models for bioreactors and of systems for clinical therapy . In vitro studies demonstrate long term external metabolic function in isolated primary hepatocytes within bioreactors . These systems are capable of supporting essential liver functions . Animal experiments verify the possibility of upscaling bioreactors for clinical treatment . However, since there is no reliable animal model for investigating the treatment of acute liver failure, the promising results obtained from these studies have limited relevance to human beings . The small number of clinical studies performed thus far are not sufficient to enable any conclusions concerning improvements in the therapy of acute liver failure . Although important progress has been made in the development of these systems, multiple hepatocyte culture models and bioreactor constructions are being discussed in the literature, indicating competition in this field of medical research . For the use of hepatocytes and sinusoidal endothelial cells in coculture, a bioreactor has been designed . The construction is based on capillaries for hepatocyte aggregate immobilization . Four separate capillary membrane systems, each permitting a different function, are woven in order to create a three-dimensional network . Cells are perfused via independent capillary membrane compartments . Decentralized oxygen supply and carbon dioxide removal with low gradients is possible . The parallel use of identical units enables easy upscaling . Initial studies on the use of discarded organs that are unsuitable for transplantation as a source for primary human liver cells seem to be promising.

Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 561 - 5
{Studies on the cell suspension culture of Saussarea medusa in a stirred tank bioreactor}; Huang Y et al.; The cell suspension culture of Saussarea medusa in a 2L aerated and agitated bioreactor with a four-pitch-blade impeller was investigated . The effects of agitation speed, aeration and inoculum size on cell growth and flavonoids production were studied and it was found that cells had optimum growth and flavonoids production when cultivated at 75 r/min, 700-1000 L/min and an inoculum of 4.0-5.0 g/L . A high cell biomass of 13.8 g/L and flavonoids production of 416 mg/L were achieved after 12 days of cultivation . Time course study revealed that flavonoids biosynthesis was growth-associated . The studies on aggregates size distribution in the bioreactor showed that the aggregates break-up caused by hydrodynamic stress might adversely affect cell growth and lead to significant reduction of cell biomass and flavonoids production.

Water Sci Technol, 2001, 44(10), 197 - 202
Sludge reduction potential of metazoa in membrane bioreactors; Luxmy BS et al.; In order to check the sludge reduction capacity of metazoa in a membrane bioreactor (MBR), pilot-scale studies were conducted . Three MBRs had been set in a wastewater treatment plant at Tokyo, Japan and they were receiving real wastewater . Initially pH inside the three MBRs was controlled as pH 7, 6 and 5 respectively . Then metazoa population was monitored along with MLSS change . It was found that the presence or absence of the metazoa population did not have any significant effect on the increasing pattern of MLSS . In the MBR with pH 6 highest accumulation of sludge was observed though a high and steady level of metazoa (1,000-2,000 per ml) was present there . But in this MBR a lot of metazoa attached in the membrane was also observed and here the increase in transmembrane pressure was less than in the other two . So, metazoa population especially the attached one in the membrane plays an effective role in fouling control of the membrane . Presence of attached media may provide a suitable niche for metazoa in the process . So, attached media known as DB lace was also inserted in MBRs for testing its capacity along with inoculum of oligochaete worms . Accumulation of sludge was not satisfactory in the attached string and it seems that inoculated worm could not adjust to the environment as they were not sludge originated . So, in the next experimental stage, attached media was inserted in the form of a bundle and this time no inoculation of worm was used . A steady metazoa population was observed in the system but the accumulation of sludge in the attached media was the same as before.

Ann Biomed Eng, 2001 Nov, 29(11), 963 - 73
Dynamic in vitro quantification of bioprosthetic heart valve leaflet motion using structured light projection; Iyengar AKS et al.; Quantification of heart valve leaflet deformation during the cardiac cycle is essential in understanding normal and pathological valvular function, as well as in the design of replacement heart valves . Due to the technical complexities involved, little work to date has been performed on dynamic valve leaflet motion . We have developed a novel experimental method utilizing a noncontacting structured laser-light projection technique to investigate dynamic leaflet motion . Using a simulated circulatory loop, a matrix of 150-200 laser light points were projected over the entire leaflet surface . To obtain unobstructed views of the leaflet surface, a stereo system of high-resolution boroscopes was used to track the light points at discrete temporal points during the cardiac cycle . The leaflet surface at each temporal point was reconstructed in three dimensions, and fit using our biquintic hermite finite element approach (Smith et al., Ann . Biomed . Eng . 26:598-611, 2001) . To demonstrate our approach, we utilized a bovine pericardial bioprosthetic heart valve, which revealed regions of complex flexural deformation and substantially different shapes during the opening and closing phases . In conclusion, the current method has high spatial and temporal resolution and can reconstruct the entire surface of the cusp simultaneously . Because it is completely noncontacting, this approach is applicable to studies of fatigue and bioreactor technology for tissue engineered heart valves.

Ann Biomed Eng, 2001 Nov, 29(11), 947 - 55
Analysis of oxygen transport to hepatocytes in a flat-plate microchannel bioreactor; Roy P et al.; Oxygen transfer to cultured hepatocytes in microchannel parallel-plate bioreactors with and without an internal membrane oxygenator was investigated with a mathematical model and the results were corroborated with fluorescence imaging experiments . The consumption of oxygen by hepatocytes was assumed to follow Michaelis-Menten kinetics . Our simulations indicate that under conditions of low Peclet (Pe) number (<80) and fixed Damlkohler number (= 0.14, corresponding to rat hepatocytes) the cells are hypoxic in the bioreactor without an internal membrane oxygenator . Under the same conditions, the bioreactor with an internal membrane oxygenator can avoid cell hypoxia by appropriate selection of membrane Sherwood number and/or the gas phase oxygen partial pressure, thus providing greater control of cell oxygenation . At high Pe, both bioreactors are well oxygenated . Experimentally determined oxygen concentrations within the bioreactors were in good qualitative agreement with model predictions . At low Pe, cell surface oxygen depletion was predicted from analytically derived criteria . Hepatocytes with oxygen dependent functional heterogeneity may exhibit optimal function in the bioreactor with the internal membrane oxygenator.

Nucleic Acids Res . 2002 Jan 15;30(2):E9.
High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells; Durocher Y et al.; A scalable transfection procedure using polyethylenimine (PEI) is described for the human embryonic kidney 293 cell line grown in suspension . Green fluorescent protein (GFP) and human placental secreted alkaline phosphatase (SEAP) were used as reporter genes to monitor transfection efficiency and productivity . Up to 75% of GFP-positive cells were obtained using linear or branched 25 kDa PEI . The 293 cell line and two genetic variants, either expressing the SV40 large T-antigen (293T) or the Epstein-Barr virus (EBV) EBNA1 protein (293E), were tested for protein expression . The highest expression level was obtained with 293E cells using the EBV oriP-containing plasmid pCEP4 . We designed the pTT vector, an oriP-based vector having an improved cytomegalovirus expression cassette . Using this vector, 10- and 3-fold increases in SEAP expression was obtained in 293E cells compared with pcDNA3.1 and pCEP4 vectors, respectively . The presence of serum had a positive effect on gene transfer and expression . Transfection of suspension-growing cells was more efficient with linear PEI and was not affected by the presence of medium conditioned for 24 h . Using the pTT vector, >20 mg/l of purified His-tagged SEAP was recovered from a 3.5 l bioreactor . Intracellular proteins were also produced at levels as high as 50 mg/l, representing up to 20% of total cell proteins.

Bioseparation, 2001, 10(1-3), 57 - 63
Capture of proteins from mammalian cells in pilot scale using different STREAMLINE adsorbents; Lutkemeyer D et al.; This presentation compares three different expanded bed matrices . STREAMLINE rProtein A, STREAMLINE SP-XL and STREAMLINE Chelating were monitored in respect to their ability to clarify the broth, to concentrate and to purify the distinct target protein . The capture of a mouse IgG1 and a recombinant prothrombin (PT) was carried out in pilot scale using a 100-l bioreactor and STREAMLINE 100 and 200 columns, respectively . The robustness of the process was also estimated monitoring the expansion behaviour and the cell and debris concentrations during the load and in the eluat . In all cases the capture of the target proteins was comparable to conventional chromatographic systems . The purification success was mainly dependent on the selectivity of the ligand used . The affinity process resulted in a highly purified product . The ion exchanger and chelating material mainly concentrated the product . In all three cases 100 l of cell broth were successfully processed in one run . The robustness of the ion exchanger process was poor, because of strong cell matrix interaction . However, for the chelating and especially for the affinity matrix a highly reproducible process was obtained.

Biotechnol Bioeng, 2002 Feb 15, 77(4), 455 - 61
Overexpression of alcohol dehydrogenase or pyruvate decarboxylase improves growth of hairy roots at reduced oxygen concentrations; Shiao TL et al.; Overexpression of Arabidopsis thaliana genes for the fermentation enzymes, alcohol dehydrogenase and pyruvate decarboxylase, improved the tolerance of A . thaliana hairy roots to low oxygen conditions . Whereas the specific growth rate of untransformed hairy roots in shake flasks and in a multiple-tube recirculation bioreactor declined significantly with decreasing oxygen tension down to 25% air saturation, growth of the transformant root lines was maintained at rates similar to those achieved with full aeration . This work demonstrates that altering the expression of selected genes involved in anaerobic metabolism can alleviate the problems of oxygen deficiency in hairy root cultures caused by poor mixing and mass transfer conditions .

Biotechnol Bioeng, 2002 Feb 15, 77(4), 381 - 93
Validation of a model for process development and scale-up of packed-bed solid-state bioreactors; Weber FJ et al.; We have validated our previously described model for scale-up of packed-bed solid-state fermenters (Weber et al., 1999) with experiments in an adiabatic 15-dm(3) packed-bed reactor, using the fungi Coniothyrium minitans and Aspergillus oryzae . Effects of temperature on respiration, growth, and sporulation of the biocontrol fungus C . minitans on hemp impregnated with a liquid medium were determined in independent experiments, and the first two effects were translated into a kinetic model, which was incorporated in the material and energy balances of the packed-bed model . Predicted temperatures corresponded well with experimental results . As predicted, large amounts of water were lost due to evaporative cooling . With hemp as support no shrinkage was observed, and temperatures could be adequately controlled, both with C . minitans and A . oryzae . In experiments with grains, strong shrinkage of the grains was expected and observed . Nevertheless, cultivation of C . minitans on oats succeeded because this fungus did not form a tight hyphal network between the grains . However, cultivation of A . oryzae failed because shrinkage combined with the strong hyphal network formed by this fungus resulted in channeling, local overheating of the bed, and very inhomogeneous growth of the fungus . For cultivation of C . minitans on oats and for cultivation of A . oryzae on wheat and hemp, no kinetic models were available . Nevertheless, the enthalpy and water balances gave accurate temperature predictions when online measurements of oxygen consumption were used as input . The current model can be improved by incorporation of (1) gas-solids water and heat transfer kinetics to account for deviations from equilibrium observed with fast-growing fungi such as A . oryzae, and (2) the dynamic response of the fungus to changes in temperature, which were neglected in the isothermal kinetic experiments .

J Endocrinol, 2002 Jan, 172(1), 199 - 210
Production and purification of recombinant human inhibin and activin; Pangas SA et al.; Inhibin and activin are protein hormones with diverse physiological roles including the regulation of pituitary FSH secretion . Like other members of the transforming growth factor-beta gene family, they undergo processing from larger precursor molecules as well as assembly into functional dimers . Isolation of inhibin and activin from natural sources can only produce limited quantities of bioactive protein . To purify large-scale quantities of recombinant human inhibin and activin, we have utilized stably transfected cell lines in self-contained bioreactors to produce protein . These cells produce approximately 200 microg/ml per day total recombinant human inhibin . Conditioned cell media can be purified through column chromatography resulting in dimeric mature 32-34 kDa inhibin A and 28 kDa activin A . The purified recombinant proteins maintain their biological activity as measured by traditional in vitro assays including the regulation of FSH in rat anterior pituitary cultures and the regulation of promoter activity of the activin-responsive promoter p3TP-luc in tissue culture cells . These proteins will be valuable for future analysis of inhibin and activin function and have been distributed to the US National Hormone and Peptide Program.

Med Clin (Barc), 2001 Dec 15, 117(20), 781 - 4
{Bioartificial liver support for acute liver failure . First case treated in Spain}; Salmeron JM et al.; BACKGROUND: Research aimed at developing artificial liver support systems has experienced a notable increase in the last decade . Hybrid systems including bioreactors containing hepatocytes which are perfused by liver failure patients blood or plasma have been deviced for the first time . The purpose of such a strategy is to substitute, at least in part, the impaired hepatic function thus improving the prognosis of patients with severe acute or chronic liver diseases . CASE REPORT: In the present paper, we report the first such a case treated in Spain in the context of a controlled, randomized, multicenter international study aimed at investigating the usefulness and safety of a bioartificial liver support system based on cryopreserved porcine hepatocytes in patients with acute liver failure or having a non-functioning primary graft after liver transplantation . RESULTS: In this first experience, two sessions of treatment could be completed before a patient with acute liver failure underwent a successful emergency liver transplantation . After more than two years of follow-up, the patient is in her normal life activities and she has not presented any adverse event related to the bioartificial liver support therapy so far . CONCLUSION: Bioartificial liver support systems are starting to be available for use in clinical practice . Yet it is mandatory to establish their safety and efficacy before a widespread recommendation.

J Orthop Res, 2001 Nov, 19(6), 1089 - 97
Integration of engineered cartilage; Obradovic B et al.; The structure and function of cartilaginous constructs, engineered in vitro using bovine articular chondrocytes, biodegradable scaffolds and bioreactors, can be modulated by the conditions and duration of tissue cultivation . We hypothesized that the integrative properties of engineered cartilage depend on developmental stage of the construct and the extracellular matrix content of adjacent cartilage, and that some aspects of integration can be studied under controlled in vitro conditions . Disc-shaped constructs (cultured for 5+/-1 days or 5+/-1 weeks) or explants (untreated or trypsin treated cartilage) were sutured into ring-shaped explants (untreated or trypsin treated cartilage) to form composites that were cultured for an additional 1-8 weeks in bioreactors and evaluated biochemically, histologically and mechanically (compressive stiffness of the central disk, adhesive strength of the integration interface) . Immature constructs had poorer mechanical properties but integrated better than either more mature constructs or cartilage explants . Integration of immature constructs involved cell proliferation and the progressive formation of cartilaginous tissue, in contrast to the integration of more mature constructs or native cartilage which involved only the secretion of extracellular matrix components. , . Integration patterns correlated with the adhesive strength of the disc-ring interface, which was markedly higher for immature constructs than for either more mature constructs or cartilage explants . Trypsin treatment of the adjacent cartilage further enhanced the integration of immature constructs.

Zhonghua Kou Qiang Yi Xue Za Zhi, 2000 Jul, 35(4), 254 - 5
{A quick way in isolation and amplification of mandibular condylar cartilage cell in vitro}; Jiao Y et al.; OBJECTIVE: To establish a quick way in acquiring well differentiated mandibular condylar cartilage (MCC) cells with high viability in large scale . METHODS: Japan white rabbit MCC cells were harvested by enzymatic method . They were grown in a modified bioreactor culture system, which contained the cytodex-3 micro-carriers in the culture medium . Kinetic growth of MCC cells on DEAE-dextran micro-carrier was observed under phase contrast microscope and environmental scanning microscope respectively . RESULTS: MCC cells attached rapidly to the surface of micro-carriers, but their spreading was slow . A quick growth of these cells was observed when they fully spread onto the micro-carrier . The number of MCC cells increased 16.2 times compared with that of plating . CONCLUSIONS: Micro-carrier culture of MCC cells can yield a large quantity of cells within a short period of time that will be of benefit in banking MCC cells for reconstruction of impaired cartilage.

Cancer Biother Radiopharm, 2001 Oct, 16(5), 381 - 90
Characterization of human tumor-infiltrating lymphocytes expanded in hollow-fiber bioreactors for immunotherapy of cancer; Malone CC et al.; We attempted to grow tumor-infiltrating lymphocytes (TIL) from 34 fresh tumors of eight different histologies using flasks for the initiation phase and hollow fiber bioreactors to expand TIL to therapeutic numbers . Overall success rate was 76% (26/34) including melanoma (9/14, 64%) and renal cell carcinoma (11/11, 100%) . The mean number of days required to reach successful initiation (1 x 10(9) TIL) for all tumor types was 29 +/- 16 days (mean +/- S.D.) . Therapeutic doses of TIL required an average of 88 +/- 23 days (initiation plus expansion) with an average TIL number of 3.2 x 10(10) +/- 2.8 x 10(10) . TIL phenotype was predominantly CD4+ in 53% (16/30) and CD8+ in 47% (14/30), renal cell carcinoma samples accounted for 12/14 of the predominantly CD8+ TIL . Cells bearing the natural killer (NK) phenotype represented only 0-7% of TIL while LAK phenotype represented 0-68% (mean 11 +/- 15%); LAK was the predominant phenotype in one patient with kidney cancer . Cytotoxicity tests showed consistent NK and LAK activity in addition to cytolysis of autologous tumor . Autologous tumor cell restricted cytolysis was noted for three TIL cultures . The overall success rate and characteristics of TIL were similar to our results with TIL expanded in semi-permeable plastic bags . This work confirms that hollow-fiber bioreactors are a suitable alternative to semi-permeable bags and roller bottle systems for the expansion of human TIL for therapeutic use in cancer patients.

FASEB J, 2002 Feb, 16(2), 270 - 2 Epub 2001 Dec 28.
Cell differentiation by mechanical stress; Altman GH et al.; Growth factors, hormones, and other regulatory molecules are traditionally required in tissue engineering studies to direct the differentiation of progenitor cells along specific lineages . We demonstrate that mechanical stimulation in vitro, without ligament-selective exogenous growth and differentiation factors, induces the differentiation of mesenchymal progenitor cells from the bone marrow into a ligament cell lineage in preference to alternative paths (i.e., bone or cartilage cell lineages) . A bioreactor was designed to permit the controlled application of ligament-like multidimensional mechanical strains (translational and rotational strain) to the undifferentiated cells embedded in a collagen gel . The application of mechanical stress over a period of 21 days up-regulated ligament fibroblast markers, including collagen types I and III and tenascin-C, fostered statistically significant cell alignment and density and resulted in the formation of oriented collagen fibers, all features characteristic of ligament cells . At the same time, no up-regulation of bone or cartilage-specific cell markers was observed.

Appl Environ Microbiol, 2002 Jan, 68(1), 173 - 80
Isolation and characterization of Novosphingobium sp . strain MT1, a dominant polychlorophenol-degrading strain in a groundwater bioremediation system; Tiirola MA et al.; A high-rate fluidized-bed bioreactor has been treating polychlorophenol-contaminated groundwater in southern Finland at 5 to 8 degrees C for over 6 years . We examined the microbial diversity of the bioreactor using three 16S ribosomal DNA (rDNA)-based methods: denaturing gradient gel electrophoresis, length heterogeneity-PCR analysis, and restriction fragment length polymorphism analysis . The molecular study revealed that the process was dependent on a stable bacterial community with low species diversity . The dominant organism, Novosphingobium sp . strain MT1, was isolated and characterized . Novosphingobium sp . strain MT1 degraded the main contaminants of the groundwater, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol, at 8 degrees C . The strain carried a homolog of the pcpB gene, coding for the pentachlorophenol-4-monooxygenase in Sphingobium chlorophenolicum . Spontaneous deletion of the pcpB gene homolog resulted in the loss of degradation ability . Phenotypic dimorphism (planktonic and sessile phenotypes), low growth rate (0.14 to 0.15 h(-1)), and low-copy-number 16S rDNA genes (single copy) were characteristic of strain MT1 and other MT1-like organisms isolated from the bioreactor.

J Chromatogr A, 2001 Dec 14, 938(1-2), 67 - 77
Recalcitrance of poly(vinylpyrrolidone): evidence through matrix-assisted laser desorption-ionization time-of-flight mass spectrometry; Trimpin S et al.; The aerobic biodegradability of an extensively used synthetic polymer was monitored the first time on a laboratory-scale fixed-bed bioreactor (FBBR) applying matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) . Polymeric poly(vinylpyrrolidone) (PVP) was spiked at concentrations of 10 mg l(-1) onto the FBBR run with river water and the biodegradation monitored after lyophilization of aliquots of the test liquor applying MALDI-TOF-MS . The latter proved to be a powerful tool for qualitative screening purposes of PVP in a molecular mass range <20 kDa in particularly yielding a high sensitivity and shot-to-shot reproducibility . The sample-to-sample reproducibility was enhanced applying the anchor target device . Post-source decay-MALDI-TOF-MS fragmentation investigations determined the unknown end groups of PVP unambiguously . Poor biodegradability of PVP can be assumed, since even after 30 days, no oxidation of the terminal groups and no difference in the repeating units was observed . A decrease in the molecular mass distribution can be drawn back rather to adsorption of PVP in the FBBR other than to biodegradation . This was further investigated performing an adsorption experiment with sewage sludge as solid matrix and analyses of the aqueous phase and sludge samples . Extrapolating these results to the situation in wastewater treatment plants, it is highly likely that PVP is eliminated from the dissolved phase by adsorption onto sludge particles.

Appl Microbiol Biotechnol, 2001 Nov, 57(4), 506 - 10
Evaluation of an electrochemical bioreactor system in the biotransformation of 6-bromo-2-tetralone to 6-bromo-2-tetralol; Shin HS et al.; Biotransformation of 6-bromo-2-tetralone (Br-beta-tetralone) to 6-bromo-2-tetralol (Br-beta-tetralol) by yeast cells of Trichosporon capitatum (ATCC 74312) and its partially purified Br-beta-tetralone reductase was evaluated in an electrochemical bioreactor . The biotransformation rates and final product formation were significantly affected by substrate concentration, biomass and electric potential . At 2 g/l of substrate, the initial reaction rate and final product were increased by 35% and 15%, respectively, with -1.5 V of electric potential compared to without electric potential . Additional substrate (2 g/l) provided by pulse feeding to the reaction mixture at different intervals resulted in 2.1 g/l Br-beta-tetralol compared to a total of 1.2 g/l without feeding . However, the increased production was not proportionate to the amount of additionally fed substrate . Increased substrate availability by the addition of 5% (v/v) ethanol resulted in the highest reaction rate and product formation, but addition of ethanol at a concentration higher than 5% decreased the reaction rate . At low biomass, the initial reaction rates were enhanced significantly when electric potential was high, but a higher biomass was necessary to obtain a similar reaction rate when electric potential was reduced . The highest initial reaction rate (59.2 mg/l per min) was achieved with a two-fold biomass concentration of 15.6 g of dry cell weight/l, substrate at 4 g/l and electric potential at -6 V . The conversion of Br-beta-tetralone to Br-beta-tetralol with partially purified Br-beta-tetralone reductase was slow in the presence of electric potential.

J Environ Sci Health B, 2001 Nov, 36(6), 821 - 34
Evaluating environmental hazards of land applying composted diazinon using earthworm bioassays; Leland JE et al.; Environmental hazards resulting from land application of composted pesticide residue have not been rigorously evaluated . This study was conducted to examine the toxicity of a composted pesticide residue using earthworms (Eisenia foetida Savigny) as a microinvertebrate model in a soil bioassay system . Diazinon, which was used in these experiments as a test pesticide, was removed from simulated rinsate (wastewater) by sorption onto peat moss . Following the rinsate clean-up phase, diazinon-laden peat moss was placed into bioreactors and composted for either 30 or 60 days . Earthworms were then exposed to soil amended with the composted material . Mortality and symptomatic effects characteristic of acetylcholinesterase inhibition, including weight loss, reduced burying ability and curling, occurred in earthworms exposed to soil amended with either uncomposted or 30-day composted diazinon, but not in those exposed to soil amended with 60-day composted diazinon . The amount of solvent-extractable diazinon from compost was not directly related to acute earthworm toxicity based on the selected criteria . These results indicated a reduction in diazinon bioavailability during latter 30 d of composting that did not correspond to a reduction in solvent-extractable diazinon concentrations . Measuring symptomatic effects of xenobiotics as described in this study may increase the sensitivity and diagnostic ability of earthworm bioassays.

J Biotechnol, 2002 Feb 28, 93(3), 269 - 82
Expression of recombinant cytoplasmic yeast pyruvate carboxylase for the improvement of the production of human erythropoietin by recombinant BHK-21 cells; Irani N et al.; Recently, a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to reconstitute the missing link between glycolysis and TCA, thus increasing the flux of glucose into the TCA and resulting in a higher intracellular ATP content . Now, these metabolically engineered cells have been additionally transfected with a plasmid bearing the gene for human erythropoietin . EPO yield and substrate-specific productivity of the recombinant BHK-21 cells have been compared to control cells without the PYC2-gene but transfected with the plasmid coding for the expression of the selection genes and EPO . PYC2-expressing clones showed a 2-fold higher glucose-specific productivity and a 2-fold higher product concentration in a continuously perfused bioreactor . Moreover, the PYC2 expression enabled the cells to become more resistant to low glucose concentrations in the culture medium . They could produce at nearly maximum productivity under glucose-limiting conditions of 0.05-1 gl(-1) that guaranteed a reduced accumulation of lactate in fed-batch production systems . Due to the fact that PYC2-expressing cells are characterized by reduced glucose consumption, a prolonged production phase in bioreactors can be maintained . Based on the demand not to fall short of 80% cell viability for the production, EPO could be produced for 2 days (30%) longer compared to the control due to a more economic exploitation of glucose, and the prolonged viability period of the cells using a batch cultivation driven by glutamine limitation.

Biotechnol Bioeng, 2002 Feb 5, 77(3), 316 - 23
On-line determination of animal cell concentration in two industrial high-density culture processes by dielectric spectroscopy; Ducommun P et al.; Dielectric spectroscopy was applied to two industrial high cell density culture processes and used to determine on-line the concentration of CHO cells immobilized on macroporous microcarriers in a stirred bioreactor and in a packed-bed of disk carriers . The cell concentration predicted from the spectroscopic data was in excellent agreement with off-line cell counting data for both processes . Deviations between the two counting methods only occurred in the case of a significant decrease of the cell viability, from 93% to 64%, which induced a change of the average cell size in the culture . Results for the packed-bed process were further confirmed by the application of indirect yield models based on the measurement of glucose, lactate, and the protein of interest . Moreover, dielectric spectroscopy was used as a tool to characterize the packed-bed process . It was possible to determine both the maximum cell concentration that could be reached in the culture system, 2.0 x 10(11) cell per kg of disk carrier, and to quantify the increase of specific protein productivity induced by the production phase, from 5.14 x 10(-8) microg x cell(-1) x h(-1) to 4.24 x 10(-7) microg x cell(-1) x h(-1) .

Biotechnol Bioeng, 2002 Jan 20, 77(2), 219 - 24
The effect of dissolved oxygen (DO) concentration on the glycosylation of recombinant protein produced by the insect cell-baculovirus expression system; Zhang F et al.; The effect of dissolved oxygen concentration on human secreted alkaline phosphatase (SEAP) glycosylation by the insect cell-baculovirus expression system was investigated in a well-controlled bioreactor . Oligomannose-type N-linked glycans (i.e., Man2 to Man6 and Man3F) were present in SEAP produced by Spodoptera frusiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines . The relative amounts of the most highly processed glycans (i.e., Man3F and Man2 in the SEAP from Sf-9 and Tn-5B1-4 cells, respectively) were significantly higher at 50% of air saturation than at either 10% or 190% of air saturation . That is, glycan processing was inhibited at both low and high dissolved oxygen concentrations .

Biotechnol Bioeng, 2002 Jan 20, 77(2), 194 - 203
Erythropoietin production from CHO cells grown by continuous culture in a fluidized-bed bioreactor; Wang MD et al.; A Chinese hamster ovary (CHO) cell line that expresses human erythropoietin (huEPO) was in a 2-L Cytopilot fluidized-bed bioreactor with 400 mL macroporous Cytoline-1 microcarriers and a variable perfusion rate of serum-free and protein-free medium for 48 days . The cell density increased to a maximum of 23 x 10(6) cells/mL, beads on day 27 . The EPO concentration increased to 600 U/mL during the early part of the culture period (on day 24) and increased further to 980 U/mL following the addition of a higher concentration of glucose and the addition of sodium butyrate . The EPO concentration was significantly higher (at least 2x than that in a controlled stirred-tank bioreactor, in a spinner flask, or in a stationary T-flask culture . The EPO accumulated to a total production of 28,000 kUnits over the whole culture period . The molecular characteristics of EPO with respect to size and pattern of glycosylation did not change with scale up . The pattern of utilization and production of 18 amino acids was similar in the Cytopilot culture to that in a stationary batch culture in a T-flask . The concentration of ammonia was maintained at a low level (< 2 mM) over the entire culture period . The specific rate of consumption of glucose, as well as the specific rates of production of lactate and ammonia, were constant throughout the culture period indicating a consistent metabolic behavior of the cells in the bioreactor . These results indicate the potential of the Cytopilot bioreactor culture system for the continuous production of a recombinant protein over several weeks .

Biotechnol Bioeng, 2002 Jan 20, 77(2), 174 - 85
Real-time in situ monitoring of freely suspended and immobilized cell cultures based on mid-infrared spectroscopic measurements; Rhiel M et al.; Glucose and lactate profiles in Chinese hamster ovary cell cultures were accurately monitored in real time and in situ during three bioreactor batch cultures lasting 11,15, and 15 days performed within a 60-day period . Monitoring was accomplished using in situ-collected mid-infrared spectra analyzed with a priori one-time established partial least-squares regression models . The robustness of the technique was demonstrated by application of these models without modification after 2.3 years . Neither recalibration nor instrument maintenance was required during the 2.3-year period, except for the daily filling of liquid nitrogen for detector cooling during operation . The lactate calibration model yielded accurate absolute concentration estimations during each of the batch cultures with standard errors of estimate from 1 to 3 mM . The a priori-established glucose calibration model yielded concentration estimations with an off-set, which was constant throughout a culture . Adjustment of the off-set before inoculation resulted in accurate concentration estimations with Standard errors of estimate of approximately 1 mM for each of the bioreactor cultures . Sensitivity in detecting differences of 0.5 mM and selectivity against variation of one metabolite while the other was kept constant was demonstrated during standard additions of either glucose or lactate . The sensor system proved to be reliable, simple, accurate, sterile, and capable of long-term automatic operation and is considered to be mature enough to be routinely applied for in situ (on-line) cell culture monitoring .

J Cell Biochem, 2001, 83(4), 574 - 84
Effect of culture in a rotating wall bioreactor on the physiology of differentiated neuron-like PC12 and SH-SY5Y cells; Wang SS et al.; A variety of evidence suggests that nervous system function is altered during microgravity, however, assessing changes in neuronal physiology during space flight is a non-trivial task . We have used a rotating wall bioreactor with a high aspect ratio vessel (HARV), which simulates the microgravity environment, to investigate the how the viability, neurite extension, and signaling of differentiated neuron-like cells changes in different culture environments . We show that culture of differentiated PC12 and SH-SY5Y cells in the simulated microgravity HARV bioreactor resulted in high cell viability, moderate neurite extension, and cell aggregation accompanied by NO production . Neurite extension was less than that seen in static cultures, suggesting that less than optimal differentiation occurs in simulated microgravity relative to normal gravity . Cells grown in a mixed vessel under normal gravity (a spinner flask) had low viability, low neurite extension, and high glutamate release . This work demonstrates the feasibility of using a rotating wall bioreactor to explore the effects of simulated microgravity on differentiation and physiology of neuron-like cells .

J Biomed Mater Res, 2002 Jan, 59(1), 127 - 35
Perfusion culture of hepatocytes within galactose-derivatized biodegradable poly(lactide-co-glycolide) scaffolds prepared by gas foaming of effervescent salts; Park TG; Galactose, a specific ligand for asialoglycoprotein receptor in hepatocytes, was immobilized onto the internal surface of highly porous biodegradable poly(D,L-lactic-co-glycolic acid) scaffolds prepared by gas foaming of effervescent salts . Rat hepatocytes seeded within the scaffolds were cultivated by using a continuous flow and perfusion reactor system . Flow rate of medium circulating through the closed loop bioreactor system was optimized to minimize the extent of cell washout from the scaffold/cell construct while satisfying the oxygen transport rate to the seeded hepatocytes . Using the flow culture system, the scaffolds immobilized with galactose onto its internal surface retained a greater number of hepatocytes than those with unmodified or immobilized with glucose due to specific interactions between seeded hepatocytes and galactose moieties exposed onto the surface of the scaffolds . The perfusion culture system based on galactose-modified macroporous scaffolds, under optimal flow conditions, resulted in much higher albumin secretion rate, approximately 70 pg/cell/day for 7 days, compared to that with glucose modified scaffolds used as a negative control. , , , , . The enhanced functional activity of hepatocytes seeded within the galactose modified scaffolds was likely caused by the formation of aggregated hepatocytes within the scaffolds .

Biotechnol Bioeng, 2002 Jan 5, 77(1), 83 - 90
A novel multi-coaxial hollow fiber bioreactor for adherent cell types . Part 1: hydrodynamic studies; Wolfe SP et al.; A novel multi-coaxial bioreactor for three-dimensional cultures of adherent cell types, such as liver, is described . It is composed of four tubes of increasing diameter placed one inside the other, creating four spatially isolated compartments . Liver acinar structure and physiological parameters are mimicked by sandwiching cells in the space between the two innermost semi-permeable tubes, or hollows fibers, and creating a radial flow of media from an outer compartment (ECC), through the cell mass compartment, and to an inner compartment (ICC) . The outermost compartment is created by gas-permeable tubing, and the housing is used to oxygenate the perfusion media to periportal levels in the ECC . Experiments were performed using distilled water to correlate the radial flow rate (Q(r)) with (1) the pressure drop (DeltaP) between the media compartments that sandwich the cell compartment and (2) the pressure in the cell compartment (P(c)) . These results were compared with the theoretical profile calculated based on the hydraulic permeability of the two innermost fibers . Phase-contrast velocity-encoded magnetic resonance imaging was used to visualize directly the axial velocities inside the bioreactor and confirm the assumptions of laminar flow and zero axial velocity at the boundaries of each compartment in the bioreactor . Axial flow rates were calculated from the magnetic resonance imaging results and were similar to the measured axial flow rates for the previously described experiments .

Biotechnol Bioeng, 2001 Dec 20, 75(6), 691 - 701
Application of redox mediators to accelerate the transformation of reactive azo dyes in anaerobic bioreactors; van der Zee FP et al.; Azo dyes are nonspecifically reduced under anaerobic conditions but the slow rates at which reactive azo dyes are converted presents a serious problem for the application of anaerobic technology as a first stage in the complete biodegradation of these compounds . As quinones have been found to catalyze reductive transfers by acting as redox mediators, the application of anthraquinone-2,6-disulfonic acid (AQDS) during continuous anaerobic treatment of the reactive azo dye, Reactive Red 2 (RR2), was evaluated . A mixture of volatile fatty acids was used as the electron-donating primary substrate . Batch experiments demonstrated that AQDS could increase the first-order rate constant of RR2 reductive cleavage by one order of magnitude . In the continuous experiment, treatment of RR2 containing synthetic wastewater in a lab-scale upflow anaerobic sludge blanket (UASB) reactor yielded low dye removal efficiencies (<30%) . Consequently, severe toxicity problems occurred, eventually resulting in almost complete inhibition of the methanogenic activity . Addition of catalytic concentrations of AQDS (19 microM) to the reactor influent caused an immediate increase in the dye removal efficiency and recovery of biological activity . Ultimately, RR2 removal efficiency stabilized at 88%, and higher AQDS loads resulted in higher RR2 removal efficiencies (up to 98% at 155 microM AQDS) . Examination of the RR2 decolorizing properties of dye-adapted reactor sludge and of nonadapted reactor seed sludge revealed that RR2 decolorization was principally a biologically driven transfer of reducing equivalents from endogenous and added substrates to the dye . Hydrogen, added in bulk, was clearly the preferred electron donor . Bacteria that couple dye decolorization to hydrogen oxidation were naturally present in seed sludge . However, enrichment was required for the utilization of electrons from volatile fatty acids for dye reduction . The stimulatory effect of AQDS on RR2 decolorization by AQDS-unadapted sludge was mainly due to assisting the electron transfer from endogenous substrates in the sludge to the dye . The stimulatory effect of AQDS on RR2 decolorization by sludge from the AQDS-exposed reactor was, in addition, strongly associated with the transfer of electrons from hydrogen and acetate to the dye, probably due to enrichment of specialized AQDS-reducing bacteria .

Biotechnol Bioeng, 2001 Dec 20, 75(6), 682 - 90
Measurement of bioreactor perfusion using dynamic contrast agent-enhanced magnetic resonance imaging; Thelwall PE et al.; Dynamic magnetic resonance imaging was used to monitor solute diffusion through aggregates of Chinese hamster ovary cells growing on macroporous carriers in a fixed-bed bioreactor . Diffusion-weighted (1)H magnetic resonance imaging (MRI) and scanning electron microscopy demonstrated that cell growth in the bioreactor was heterogeneous, with the highest cell densities being found at the periphery of the carriers . T(1)-weighted magnetic resonance imaging measurements of the inflow of a commonly used magnetic resonance contrast agent, gadolinium-diethylenetriaminopentaacetic acid (Gd-DTPA), showed that migration of the agent through the peripheral cell masses could be explained by diffusion . However, appearance of the contrast agent in the center of the carriers was too fast to be explained by simple diffusion and indicated that these regions were perfused by convective flow . The average diffusivity of Gd-DTPA through the cell mass was found to be (2.4 +/- 0.2) x 10(-10) m(2) sec(-) (mean +/- SEM) . This technique will be useful in the characterization and development of high-cell-density bioreactor systems, in which solute transport plays a critical role in cell growth and physiology .

J Hazard Mater, 2002 Jan 28, 89(2-3), 241 - 52
A pilot study for the selection of a bioreactor for remediation of groundwater from a coal tar contaminated site; Guerin TF; Coal tars in soil at a gasworks site in South Eastern Australia led to groundwater contamination with polycyclic aromatic hydrocarbons (PAHs), mono-aromatic compounds (BTEX) and phenols . The scope of the study included testwork in laboratory scale bioreactors and evaluation of available commercial groundwater treatment units . Two bioreactor configurations, a submerged fixed film reactor (SFFR) and a fluidized bed bioreactor (FBR) were effective, with high efficiencies of contaminant removal (typically >90%) over a range of hydraulic retention times (HRT) (3-29 h) . Specifically, concentrations of total PAH, naphthalene, pyrene and total phenols in the feedstock and effluent of the SFFR were 123, 60, 51, 1.38 and 0.004, 0.001, 0.004, 0.1mg/l, respectively . The FBR was only marginally less effective than the SFFR for the same groundwater contaminants . Discharge to sewer was the most appropriate end use for the effluent . SFFRs are regarded as being simpler in design and operation, and a commercially available unit has been identified which would be suitable for treating small volumes (<10 m(3) per day) of contaminated water collected at an interception trench at the site.

J Biotechnol, 2002 Feb 14, 93(2), 109 - 19
Bioactive derivatives from oleuropein by a biotransformation on Olea europaea leaf extracts; Briante R et al.; A very simple method is proposed to produce, using non-homogeneous hyperthermophilic beta-glycosidase immobilised on chitosan, 3,4-dihydroxy-phenylethanol (hydroxytyrosol), a commercially unavailable compound with well known biological properties which justify a potential commercial application . Leaf extracts from Olea europaea with high oleuropein content are selected as substrate for biotransformation . Under the biotransformation conditions, high amounts of hydroxytyrosol are collected within a short space of time after being preliminarily purified by a non-treated chitosan column . This is possible due to the capacity of amino groups on the chitosan to bind aldehydic groups of molecules present at the end of the reaction . We have produced a natural and non-toxic product from vegetal source, as opposed to the molecule obtainable through chemical synthesis, as a candidate to test in vivo its biological properties . The proposed process may prove useful for a further application for recycling Olea europaea leaves . The radical-scavenging properties of the bioreactor eluates and their capacity to inhibit fatty acid peroxidation rates are characterized in order to make them candidates as substitutes for synthetic antioxidants commonly used to increase the shelf-life of food products as well as for their possible protective effect in human cells.

Biotechnol Prog, 2001 Nov-Dec, 17(6), 1042 - 8
Effect of oscillating dissolved oxygen tension on the production of alginate by Azotobacter vinelandii; Trujillo-Roldan MA et al.; The effect of oscillating dissolved oxygen tension (DOT) on the metabolism of an exopolysaccharide-producing bacteria (Azotobacter vinelandii) was investigated, particularly on the mean molecular weight (MMW) of the alginate produced . Sinusoidal DOT oscillations were attained by manipulating the oxygen and nitrogen partial pressures at the inlet of a 1.0 L working volume bioreactor . Periods of 1200, 2400, and 4000 s and average amplitudes between 1.0% and 2.2% DOT, with an oscillation axis fixed at 3% DOT, were tested . A culture carried out at constant 3% DOT was used as comparison . The average wave amplitude had an important effect on the maximum mean molecular weight (MMW(max)) of the alginate produced . The higher the amplitude, the lower the MMW(max) . As the average wave amplitudes decreased from 2.2% to 1.0%, the MMW(max) increased from 64 to 240 KDa, respectively . Furthermore, at 3% constant DOT (0.0% of amplitude), a MMW(max) of 350 KDa was obtained . No important effect of the oscillating DOT on kinetics of biomass growth, alginate production, and sucrose consumption was observed, compared with constant DOT . The findings of this study point out that accurate DOT control is crucial if a particular molecular weight species of alginate needs to be produced, particularly in large fermentors, where bacteria are exposed to an oscillatory environment as a result of DOT gradients caused by the high viscosity of the broth and insufficient mixing.

Water Sci Technol, 2001, 44(8), 67 - 76
New developments in reactor and process technology for sulfate reduction; Pol LW et al.; Sulfate reduction has been regarded in the past as an unwanted process in anaerobic treatment of sulfate-rich wastewaters . Research efforts were primarily focused on H2S toxicity, competition between sulfidogenic and methanogenic microorganisms and suppression of sulfidogenesis . More recently, the potential sulfidogenesis for treating a wide range of wastestreams contaminated with oxidized sulfurous compounds and/or heavy metals was also appreciated . Heavy metals can be removed by the formation and subsequent precipitation of poorly soluble metal sulfides . Basically two approaches can be distinguished in wastewater treatment: passive treatment using low-cost technologies and active treatment in newly developed bioreactors . Both strategies are discussed.

Water Sci Technol, 2001, 44(8), 57 - 66
The biological sulfur cycle: novel opportunities for environmental biotechnology; Lens PN et al.; Although the study of sulfur cycle bacteria was already started around the 1890s by the famous microbiologists Winogradsky and Beijerinck, there are nowadays still many new discoveries to be made about the metabolic properties, phylogenetic position and ecological behaviour of bacteria that play a role in the biological sulfur cycle . The current interest of the scientific community in the biological sulfur cycle is very high, especially because of the many special organisms that have recently been discovered in the deep sea and other environments characterised by extreme conditions (such as high salt, low/high pH or temperature) and also in bioreactor environments . This paper highlights some of these new discoveries and relates them to environmental biotechnology . It is concluded that the micro-organisms from the sulfur cycle offer unique opportunities for sulfur pollution abatement and sulfur recovery.

Adv Biochem Eng Biotechnol, 2001, 72, 63 - 102
Integrated bioprocessing for plant cell cultures; Choi JW et al.; Plant cell suspension culture has become the focus of much attention as a tool for the production of secondary metabolites including paclitaxel, a well-known anticancer agent . Recently, it has also been regarded as one of the host systems for the production of recombinant proteins . In order to produce phytochemicals using plant cell cultures, efficient processes must be developed with adequate bioreactor design . Most of the plant secondary metabolites are toxic to cells at the high concentrations required during culture . Therefore, if the product could be removed in situ during culture, productivity might be enhanced due to the alleviation of this toxicity . In situ removal or extractive bioconversion of such products can be performed by in situ extraction with various kinds of organic solvents . In situ adsorption using polymeric resins is another possibility . Using the fact that secondary metabolites are generally hydrophobic, various integrated bioprocessing techniques can be designed not only to lower toxicity, but also to enhance productivity . In this article, in situ extraction, in situ adsorption, utilization of cyclodextrins, and the application of aqueous two-phase systems in plant cell cultures are reviewed.

Adv Biochem Eng Biotechnol, 2001, 72, 157 - 82
Large-scale plant micropropagation; Honda H et al.; Plant micropropagation is an efficient method of propagating disease-free, genetically uniform and massive amounts of plants in vitro . The micropropagation from cells can be achieved by direct organogenesis from hairy roots or regeneration via somatic tissue . Once the availability of embryogenic cell and hairy root systems based on liquid media has been demonstrated, the scale-up of the whole process should be established by an economically feasible technology for their large-scale production in appropriate bioreactors . It is necessary to design a suitable bioreactor configuration that can provide adequate mixing and mass transfer while minimizing the intensity of shear stress and hydrodynamic pressure . Automatic selection of embryogenic calli and regenerated plantlets using an image analysis procedure should be associated with the system . Using the above systems, it will be possible to establish an advanced plant micropropagation system in which the plantlets can be propagated without soil under optimal conditions controlled in plant factory . The aim of this review is to identify the problems related to large-scale plant micropropagation via somatic embryogenesis and hairy roots, and to summarize the most recent developments in bioreactor design . Emphasis is placed on micropropagation technology and computer-aided image analysis, including the successful results obtained in our laboratories.

Adv Biochem Eng Biotechnol, 2001, 72, 1 - 26
Biochemical engineering of the production of plant-specific secondary metabolites by cell suspension cultures; Zhong JJ; Plant cell culture has recently received much attention as a useful technology for the production of valuable plant-derived secondary metabolites such as paclitaxel and ginseng saponin . The numerous problems that yet bewilder the optimization and scale-up of this process have not been over emphasized . In spite of the great progress recorded in recent years towards the selection, design and optimization of bioreactor hardware, manipulation of environmental factors such as medium components, light irradiation, shear stress and O2 supply needs detailed investigations for each case . Recent advances in plant cell processes, including high-density suspension cultivation, continuous culture, process monitoring, modeling and control and scale-up, are also reviewed in this chapter . Further developments in bioreactor cultivation processes and in metabolic engineering of plant cells for metabolite production are expected in the near future.

Free Radic Biol Med, 2001 Dec 1, 31(11), 1388 - 95
The stomach as a bioreactor: dietary lipid peroxidation in the gastric fluid and the effects of plant-derived antioxidants; Kanner J et al.; Atherosclerosis may result partly from processes that occur following food consumption and that involve oxidized lipids in chylomicrons . We investigated reactions that could occur in the acidic pH of the stomach and accelerate the generation of lipid hydroperoxides and co-oxidation of dietary constituents . The ability of dietary polyphenols to invert catalysis from pro-oxidation to antioxidation was examined . The acidic pH of gastric fluid amplified lipid peroxidation catalyzed by metmyoglobin or iron ions . Metmyoglobin catalyzed peroxidation of edible oil, resulting in 8-fold increase of hydroperoxide concentration . The incubation of heated muscle tissue in simulated gastric fluid for 2 h enhanced hydroperoxides accumulation by 6-fold to 1200 microM . In the presence of catechin or red wine polyphenols, metmyoglobin catalyzed the breakdown of hydroperoxides to zero, totally preventing lipid peroxidation and beta-carotene cooxidation . We suggest that human gastric fluid may be an excellent medium for enhancing the oxidation of lipids and other dietary constituents . The results indicate the potentially harmful effects of oxidized fats intake in the presence of endogenous catalysts found in foods, and the major benefit of including in the meal plant dietary antioxidants.

Life Support Biosph Sci, 2001, 8(1), 23 - 31
Electrochemical removal of ammonium ions from a bioreactor effluent; Kim J et al.; Ammonium ions are byproducts of the oxidation of nitrogen-containing substances occurring in the initial treatment steps of water recovery systems . Removal of ammonium ions from the effluent stream from 1000 ppm to less than 0.25 ppm is an imperative need as a part of the space life support infrastructure . Drawbacks associated with processes proposed in the past include the generation of a secondary waste, cost, size, and/or the use of consumables that need to be stored or supplied . Lynntech has developed a technology that is based on an innovative, environmentally friendly electrochemical process for the effective removal of ammonium ions . The process does not use consumables except for oxygen gas from air, and does not generate a secondary waste . By controlling operational conditions, the ammonium ions may be transformed to nitrogen gas and/or to nitrate ions . Other advantages of the process include: it is energy efficient, operates at room temperature, and is microgravity compatible . Grant numbers: NAS9-00013.

J Environ Sci (China), 2001 Oct, 13(4), 508 - 13
Effect of leachate recycle and inoculation on microbial characteristics of municipal refuse in landfill bioreactors; Shen DS et al.; Population development of key groups of anaerobic and aerobic bacteria involved in municipal refuse decomposition under laboratory landfill bioreactors with and without leachate recycle and inoculation was measured since modeling municipal refuse was landfilled in bioreactors for about 210 days . Hydrolytic fermentative bacteria (HFB), hydrogen-producing acetogenic bacteria (HPAB), methane-producing bacteria (MPB), sulfate-reducing bacteria (SRB), anaerobic and aerobic cellulolytic bacteria and denitrabacteria were enumerated by the most probable number technique . The results showed that the dominant microorganism groups were the methanogenic bacteria including hydrolytic fermentative, hydrogen-producing acetogenic and methane-producing bacteria . They were present in fresh refuse but at low values and positively affected by leachate recycle and refuse inoculation . The amounts of HFB or HPAB in digesters D4 and D5 operated with inoculation and leachate recycle reached their maximum values of 10(10)-10(12) cells/g dry refuse for HFB or 10(5)-10(6) cells/g dry refuse for HPAB on day 60, in digester D3 operated with leachate recycle on day 120 for HFB (10(9) cells/g dry refuse) or on day 90 for HPAB (10(5) cells/g dry refuse), and in digesters D1 and D2 on day 210 for HFB (10(9) cells/g dry refuse) or on day 90 for HPAB (10(4)-10(6) cells/g dry refuse) . The population of methane-producing bacteria in digesters D4 and D5 sharply increased on days 60 and 90 respectively, however in digesters D1, D2 and D3 on day 120 . Leachate recycle and inoculation changed the cellulolytic microorganisms composition of refuse ecosystem, the higher amounts of anaerobic cellulolytic bacteria were measured in digesters D4 and D5 (10(7) cells/g dry refuse), followed by digesters D3 (10(6) cells/g dry refuse), D2 or D1(10(4) cells/g dry refuse) . However, the amounts of aerobic cellulolytic bacteria were much lower than that of anaerobic cellulolytic bacteria . And it was higher in digester D3 than those in digesters D1, D2, D4 and D5. , , , , . The amounts of SRB and denitrabacteria were also higher in digester D5 than those in digesters D1, D2, D3 and D4 . Refuse decomposition could be accelerated by leachate recycle and inoculation in the view of microorganism development.

Artif Organs, 2001 Sep, 25(9), 740 - 8
Long-term expression of highly differentiated functions by isolated porcine hepatocytes perfused in a radial-flow bioreactor; Morsiani E et al.; To overcome the limitations of standard hollow-fiber module in ensuring efficient cell perfusion and long-term expression of highly differentiated hepatocyte functions, we developed a novel bioreactor in which a three-dimensional hepatocyte culture system was perfused in radial-flow geometry . Isolated porcine hepatocytes were cultured for 2 weeks in recirculating serum-free tissue culture medium, in which NH4Cl and lidocaine were repeatedly added, and ammonia removal, urea synthesis, monoethylglycinexylide (MEGX) production, albumin secretion, Po2, Pco2, O2 consumption, and pH were measured thereafter . During the whole duration of the study, ammonia removal was paralleled by urea production, while MEGX concentration was constantly increased . Our results indicated that hepatocytes remained differentiated and metabolically active throughout the duration of the study . The radial-flow bioreactor allowed physiological contact between recirculating fluid and cells by equalizing the concentration of the perfusing components, including O2, throughout the module, suggesting a potential use of this configuration for extracorporeal liver support.

Artif Organs, 1994 Aug, 18(8), 611 - 7
Culture of a differentiated liver cell line, Hep G2, in serum with application to a bioartificial liver: effect of supplementation of serum with amino acids; Melkonian AD et al.; Much effort has been directed toward the development of serum-free, hormonally defined culture conditions for the maintenance of differentiated functions in many cell types, including hepatocytes . However, in the development of a hepatocyte bioreactor for artificial liver support, many designs propose the maintenance of cells in plasma as opposed to defined culture medium . There is very little reported literature on the growth and function of cells cultured in plasma or serum; therefore, the effect of increasing serum concentrations was investigated using the human hepatoma, Hep G2, as a model cell line . It was found that Hep G2 can survive and grow in 100% serum if the serum is supplemented with L-glutamic acid, glycine, and L-cysteine.

Anal Biochem, 1994 Aug 1, 220(2), 297 - 302
On-line enzymatic amplification by substrate cycling in a dual bioreactor with rotation and amperometric detection; Raba J et al.; The amplification approach centered on the cycling of two reversibly interconvertible chemical species sequentially participating in two different enzyme-catalyzed reactions (enzymatic amplification by substrate cycling) has been implemented on-line into a continuous-flow/stopped-flow/continuous-flow operation . The implementation is illustrated with the determination of L-lactate in a dual enzyme reactor containing immobilized lactate oxidase (LOD, EC 1.1.3.x) to catalyze the oxidation of L-lactate by dissolved oxygen . The immobilized LOD was affixed to a rotating disk in the lower part of the flow-through cell . Immobilized lactate dehydrogenase (EC 1.1.1.27), affixed to the top part of the cell regenerates L-lactate with the mediation of beta-NADH as the hydrogen donor . The substrate cycling permits the generation of H2O2 beyond the stoichiometric limitation, and this is detected at a stationary Pt-ring electrode located at the bottom part of the cell . The stationary Pt-ring electrode is positioned concentrically to the rotating disk containing the immobilized LOD . The resulting amplified response permits, in a simple manner, achievement of detection limits as low as 0.3 fmol.liter-1 and allows the processing of 30 samples per hour.

Biotechnol Appl Biochem, 1994 Aug, 20 ( Pt 1), 79 - 92
Kinetic study of anaerobic digestion of fruit-processing wastewater in immobilized-cell bioreactors; Borja R et al.; The kinetics of the anaerobic digestion of a fruit-processing wastewater {chemical oxygen demand (COD) = 5.1 g/l} were investigated . Laboratory experiments were carried out in bioreactors containing supports of different chemical composition and features, namely bentonite and zeolite (aluminum silicates), sepiolite and saponite (magnesium silicates) and polyurethane foam, to which the microorganisms responsible for the process adhered . The influence of the support medium on the kinetics was compared with a control digester with suspended biomass . Assuming the overall anaerobic digestion process conforms to first-order kinetics, the specific rate constant, K0, was determined for each of the experimental reactors . The average values obtained were: 0.080 h-1 (bentonite); 0.103 h-1 (zeolite); 0.180 h-1 (sepiolite); 0.198 h-1 (saponite); 0.131 h-1 (polyurethane); and 0.037 h-1 (control) . The results indicate that the support used to immobilize the micro-organisms had a marked influence on the digestion process; the results were significant at the 95% confidence level . Methanogenic activity increased linearly with COD, with the saponite and sepiolite supports showing the highest values . The yield coefficient of methane was 270 ml of methane (under standard temperature and pressure conditions)/g of COD . The average elimination of COD was 89.5%.

J Biotechnol, 1994 Jul 29, 36(1), 35 - 8
A comparison of monoclonal antibody productivity in different hollow fiber bioreactors; Lowrey D et al.; Cell culture in hollow fiber bioreactors has been used as a method for large-scale production of monoclonal antibodies, viruses, cell-associated proteins and cancer antigens . We have examined an important variable in culturing cells in hollow fiber bioreactors: bioreactor composition . Eight different bioreactor designs which varied in nominal molecular weight cutoff, surface area, fiber material and ultra filtration rate were compared in large-scale hollow fiber cultures . A standard protocol utilizing the hybridoma 3C11 (ATCC HB 8511) or African green monkey kidney cells (Vero cells, ATCC CRL 1587) was designed so that the only variable examined was the hollow fiber bioreactor in use . The results suggest that surface area has little effect on antibody productivity, while fiber composition and ultrafiltration rate may play an important role . Vero cell growth was affected by both fiber composition and ultrafiltration rate.

Int J Biochem, 1994 Jul, 26(7), 859 - 70
Transgenic bioreactors; Janne J et al.; 1 . Although many human therapeutic proteins are currently produced in microbial fermentors using recombinant DNA techniques, it is obvious that microbial processing is not suitable for a large number of bioactive proteins owing to the inability of bacteria to carry out postsynthetic modification reactions required for full biological activity . 2 . This disadvantage does not apply to animal cell bioreactors that can generate biologically fully active entities, yet the use of large-scale animal cell cultures for production purposes is prohibitively expensive . 3 . With the advent of transgenic technology, the production of valuable human pharmaceuticals in large farm animals (pig, sheep, goat and dairy cattle) has become more and more attractive as a high-quantity, low-cost alternative . By employing targeted gene transfer, e.g . using mammary gland-specific regulatory sequences fused with the desired production genes, it is possible to govern the expression to occur exclusively in the mammary gland and hence the gene product is being ultimately secreted in the milk . 4 . While reviewing the remarkable progress in this field that has even led to commercial exploitations, we will outline in somewhat greater detail our strategy for the use of dairy cattle as a bioreactor for valuable proteins of pharmaceutical interest.

Am J Physiol, 1994 Jul, 267(1 Pt 1), C195 - 203
31P-MRS measurements of extracellular pH of tumors using 3-aminopropylphosphonate; Gillies RJ et al.; The extracellular pH (pHex) of tumors is generally acidic . However, it is only recently that noninvasive magnetic resonance spectroscopic (MRS) measurements have determined that the intracellular pH (pHin) of tumor cells in situ is neutral or slightly alkaline compared with that of normal tissues . Thus cells in tumors maintain larger pH gradients than do cells in nontumor tissues . To date, measurements of pHex in tumors have been made using microelectrodes, which preclude measurement of pHex and pHin within the same preparation . In addition, microelectrodes are invasive and have the potential to alter the measured pH values . The present communication describes simultaneous measurement of pHex and pHin in vitro in bioreactor culture and in vivo using 31P-MRS analyses of 3-aminopropylphosphonate (3-APP) and inorganic phosphate . In vitro results indicate that 3-APP is not toxic and that its resonant frequency is sensitive to pH and not significantly affected by temperature or ionic strength . Bioreactor experiments indicate that this compound is neither internalized nor metabolized by cells . Experiments in vivo indicate that 3-APP can be administered intraperitoneally and that RIF-1 tumors maintain a steady-state pHin of 7.25 and a pHex of 6.66 . These data have significance to basic tumor cell physiology and to the design of approaches to cancer chemotherapy and hyperthermic therapy, because both of these modalities exhibit pH sensitivity . It is also likely that these techniques will be applicable to localized MRS of other organ systems in vivo.

Cell Transplant, 1994 Jul-Aug, 3(4), 263 - 71
Expansion of activated lymphocytes obtained from renal cell carcinoma in an automated hollow fiber bioreactor; Hillman GG et al.; Immunotherapy using IL-2 alone or combined with activated lymphocytes has been promising for metastatic renal cell carcinoma . Cytotoxic lymphocytes can be isolated from tumors, expanded in vitro with IL-2, and adoptively transferred back into the tumor-bearing host . These cells can also be transduced with the genes coding for cytokines for local delivery to tumor sites . A major drawback in adoptive immunotherapy is the cumbersome and expensive culture technology associated with the growth of large numbers of cells required for their therapeutic effect . To reduce the cost, resources, and manpower, we have developed the methodology for lymphocyte activation and expansion in the automated hollow fiber bioreactor IMMUNO*STAR Cell Expander (ACT BIOMEDICAL, INC) . Tumor Infiltrating Lymphocytes (TIL) isolated from human renal cell carcinoma tumor specimens were inoculated at a number of 10(8) cells in a small bioreactor of 30 ml extracapillary space volume . We have determined the medium flow rates and culture conditions to obtain a significant and repeated expansion of TIL at weekly intervals . The lymphocytes cultured in the bioreactor demonstrated the same phenotype and cytotoxic activity as those expanded in parallel in tissue culture plates . Lymphocyte expansion in the hollow fiber bioreactor required lower volumes of medium, human serum, IL-2 and minimal labor . This technology may facilitate the use of adoptive immunotherapy for the treatment of refractory malignancies.

Braz J Med Biol Res, 1994 Jul, 27(7), 1575 - 87
Studies on the efficiency of measles virus antigen production using VERO cell culture in a microcarrier system; Mendonca RZ et al.; 1 . A large amount of antigen is required to conduct seroepidemiologic surveys of measles . Thus, a process to obtain measles virus antigen using a bioreactor was standardized . 2 . The virus was grown in a 3.7-1 culture of VERO cells using a Celligen cell culture system containing 2 mg/ml of microcarriers (cytodex I) at 37 degrees C and 60 rpm . The cultures infected with 0.5 m.o.i . of measles virus were harvested after the appearance of the cytopathic effect . The virus suspension was clarified and concentrated by ultracentrifugation . Intracellular and extracellular virus titers were determined by hemagglutination (HA) and by induction of a cytopathic effect in cell culture (TCID50) . 3 . Intracellular virus presented 5-7 x 10(6) TCID50/0.1 ml, HA activity per 50 microliters equal to 32, with a total HA activity of 4,480 HA units (HAU) and specific activity of 116 HAU/mg . In the concentrated supernatants, the HA titer of extracellular virus was 64, with a total HA activity of 1,024 HAU and a specific activity of 1,600 HAU/mg . 4 . The antigen obtained was suitable for the detection of antibodies against measles virus in assays such as ELISA and DOT-ELISA (using 1 micrograms/well to ELISA and 2 micrograms/DOT) . 5 . The microcarrier system produced antigen sufficient for 26 ELISAs/ml compared to 5.7 ELISAs/ml obtained for the static culture system.

Biotechnol Prog, 1994 Jul-Aug, 10(4), 410 - 20
Cultivation of recombinant baby hamster kidney cells in a fluidized bed bioreactor system with porous borosilicate glass; Kratje RB et al.; Dense cell cultivation of the recombinant cell line BHK 21 pSVIL2 was performed in a fluidized bed bioreactor system containing porous borosilicate glass carriers . Experiments were carried out with different medium formulations for a period of 48 days . Due to an effective immobilization of the cells in the reactor, continuous operation was easy to perform . Maximal cell densities and product yields could be maintained, even when protein-free medium was perfused exceeding 2 reactor volumes per day . Final cell densities of magnitude 7.1 x 10(7) mL-1 intrasphere volume were reached, while the interleukin-2 production rate was 0.70 mg day-1 . The cell specific productivity reached a value of 1.3 x 10(-10) mg day-1 . The first results were presented with a cell line that grows under glutamine-free medium conditions . The use of a glutamine-free medium for the cultivation of the cells resulted in a drastic decrease in cell metabolism . Furthermore, the amino acids lysine and histidine were produced and secreted into the culture supernatant, although these metabolites normally are considered to be essential for animal cells grown in vitro . However, no lethal effect on the cells has been detected, and the total number of cells in the reactor remained constant . The metabolism of threonine has been detected to be directly dependent on the presence of glutamine . Cells grown in glutamine-free culture medium produced glycine yields 6 times higher than those grown in glutamine-containing medium . A bead-to-bead transfer of the cells has also been detected when the cells immobilized within the intrasphere volume of the borosilicate carriers reached the stationary phase.

Appl Microbiol Biotechnol, 1994 Jul, 41(5), 578 - 83
Overproduction of mannitol dehydrogenase in Rhodobacter sphaeroides; Schneider KH et al.; Mannitol dehydrogenase (MDH) from Rhodobacter sphaeroides Si4 was overproduced by constructing a strain that overexpresses the MDH gene and by producing high cell concentrations via fed-batch cultivation in a bioreactor . With the gene of mannitol dehydrogenase (mtlK) cloned into the expression vector pKK223-3 expression of MDH in Escherichia coli was obtained, but the specific enzyme activity was lower than in R . sphaeroides Si4 . In order to overexpress mtlK in R . sphaeroides, plasmid pAK82 was constructed by cloning a DNA fragment carrying mtlK into the broad-host-range expression vector pRK415 . When pAK82 was introduced into R . sphaeroides Si4 the specific mannitol dehydrogenase activity in the strain obtained was 0.48 unit (U)mg-1,3.4-fold higher than in the wild type . In this way the enzyme yield from cultivation in a bioreactor could be improved from 110 Ul-1 to 350 Ul-1 . A further increase in productivity was obtained by fed-batch cultivation of R . sphaeroides Si4 {pAK82} . Using this cultivation method an optical density of 27.6 was reached in the bioreactor, corresponding to a dry mass of 16.6 g l-1 . Since MDH formation correlated with biomass production, the MDH yield could be raised to 918 Ul-1, an 8.3-fold increase in comparison to batch cultivation of the wild-type strain.

J Chem Technol Biotechnol, 1994 Jul, 60(3), 327 - 34
Kinetics of anaerobic digestion of soft drink wastewater in immobilized cell bioreactors; Borja R et al.; A kinetic study of the anaerobic digestion of soft drink wastewater was undertaken, using bioreactors containing various suspended supports (bentonite, zeolite, sepiolite, saponite and polyurethane foam), on to which the microorganisms effecting the purification were immobilized . Assuming the overall anaerobic digestion process conforms to first-order kinetics, the specific rate constants, K0, derived from the reactors with saponite and sepiolite (magnesium silicates) were approximately twice those from bentonite and zeolite (aluminium silicates) and almost five times higher than in the control reactor (without support); the polyurethane support showed an intermediate behaviour . The methanogenic activity increased linearly with COD load, with saponite and sepiolite supports showing the highest values . The average yield coefficient of methane was 325 cm3 CH4 STP g-1 COD and the percentage elimination of COD was 77.8%; these values were not significantly altered by the type of support used.

J Biotechnol, 1994 Jun 15, 35(1), 1 - 7
Large-scale cultivation of Catharanthus roseus cells: production of ajmalicine in a 20-l airlift bioreactor; Fulzele DP et al.; Bioreactor systems have been developed for the production of ajmalicine, an alkaloid used in the treatment of hypertension . Cell cultures of Catharanthus roseus produced higher levels of ajmalicine (323 micrograms g-1 dry weight) in a production medium enriched with tryptophan . The cell cultures were grown in medium prepared in tap water and market sugar with a view to minimise the costs of production . Large-scale cultivation of cell suspension was performed in a 20-l airlift bioreactor under controlled conditions . An ajmalicine production of 315 micrograms g-1 dry weight was achieved in the bioreactor after 14 d of cultivation.

Chin Med Sci J, 1994 Jun, 9(2), 71 - 4
High density cultivation of genetically-engineered CHO cell lines with microcarrier culture systems; Xiao C et al.; Genetically-engineered CHO cell lines, r beta-13 and CLF-8B2, were cultivated with the MC-1 microcarrier culture system . The cell density could be enhanced by increasing the concentration of microcarrier . At a microcarrier concentration of 10 mg/ml, the cell density could reach 4 to 5 x 10(6) cells/ml . It was shown that these cell lines would spontaneously release from the microcarrier to attach to and proliferate on fresh microcarriers . We were thus able to scale up cultivation using a simple method, i.e . by adding fresh microcarriers and medium directly into the culture system to about 2, 4 or 8 times the original volume . Using a perfusion culture system, we have successfully cultivated CLF-8B2 cells in a 2 L bioreactor for several weeks at medium perfusion rates of 0.5 to 3 working volumes . Prourokinase was stably secreted.

Enzyme Microb Technol, 1994 Jun, 16(6), 506 - 12
Optimization of the growth conditions of Sf21 insect cells for high-density perfusion culture in stirred-tank bioreactors; Deutschmann SM et al.; Spodoptera frugiperda insect cells (IPLB-Sf21-AE) (Sf21), infected with baculovirus expression vectors during their exponential growth phase, are commonly used to produce a variety of heterologous recombinant proteins . In the present study the culture conditions of these insect cells were studied to establish high-density suspension cultures with prolonged exponential growth phases . The Sf21 cells were grown in 125-ml spinner flasks using five different culture media supplemented with 5% fetal calf serum and four protein-free or low-protein culture media . The best results were achieved in EX-CELL 401 (protein-free media) and in IPL-41 modified with 2.5 g l-1 tryptose phosphate broth (serum-supplemented media), respectively . The latter was used for further batch and continuous cultivation of Sf21 cells in a perfused 1.4-l stirred-tank bioreactor with special attention to the oxygen requirement of these cells. , , , , . Optimal growth was found at an oxygen concentration of 70% air saturation, resulting in a prolonged exponential growth phase that could be maintained for more than 16 days . A maximum cell density of 5.5 x 10(7) viable cells ml-1 was achieved.

Hum Cell, 1994 Jun, 7(2), 95 - 100
{A new liver support system composed of functional human cells and a radial-flow bioreactor}; Kawada M et al.; An artificial liver will be useful for the treatment of acute hepatic failure and a bridge of liver transplantation . The current reports suggest that the hybrid type of artificial liver composed of functional human liver cells and a bioreactor is practical for clinical use . In the present study, we succeeded high density culture on a large-scale of human functional hepatoma (JHH-7) using a newly developed radial flow packed-bed bioreactor . Since the shear stress of this bioreactor is lower than the other type, high density culture without cell damage is possible . JHH-7 cells produced large amounts of human albumin and other liver specific proteins, and then have the function of ammonia metabolism in the system . This study suggests that a radial flow bioreactor will be developed as a new type of artificial liver.

J Biotechnol, 1994 May 31, 34(3), 247 - 57
Re-use of spent cell culture medium in pilot scale and rapid preparative purification with membrane chromatography; Riese U et al.; Based on experiments in bench scale, a recycling of spent cell culture medium was performed in a 100-1 pilot scale bioreactor . The cell cultivation has been done as a repeated batch procedure after the initial batch in the following four repeated batches spent medium from the previous batch was partially re-used . After microfiltration and ultrafiltration a part of the filtrate was mixed with a concentrate of amino acids and glucose, sterile filtered and subsequently filled back into the bioreactor . Up to 65% of the harvested cell- and product-free spent medium was re-used in each repeated batch . This procedure results in a saving of pure and waste water volume and saving of supplemented proteins as transferrin, insulin and lipoproteins and, therefore, also in a reduction of the production costs . A strongly acidic membrane ion exchanger was evaluated for the ability to purify the monoclonal antibodies from the pilot scale cultivation . Within minutes, gram quantities of product could be purified in a high flux system, especially developed for this purpose, achieving purities of 80% . The capacity of the acidic membrane ion exchanger was found in former investigations to be 1 mg cm-2 with recoveries up to 96% . Final purification was carried out by gel column filtration.

Nature, 1994 May 26, 369(6478), 298 - 301
Synthesis of polymeric microcapsule arrays and their use for enzyme immobilization; Parthasarathy RV et al.; Current methods for immobilizing enzymes for use in bioreactors and biosensors include adsorption on or covalent attachment to a support, micro-encapsulation, and entrapment within a membrane/film or gel . The ideal immobilization method should employ mild chemical conditions, allow for large quantities of enzyme to be immobilized, provide a large surface area for enzyme-substrate contact within a small total volume, minimize barriers to mass transport of substrate and product, and provide a chemically and mechanically robust system . Here we describe a method for enzyme immobilization that satisfies all of these criteria . We have developed a template-based synthetic method that yields hollow polymeric microcapsules of uniform diameter and length . These microcapsules are arranged in a high-density array in which the individual capsules protrude from a surface like the bristles of a brush . We have developed procedures for filling these microcapsules with high concentrations of enzymes . The enzyme-loaded microcapsule arrays function as enzymatic bioreactors in both aqueous solution and organic solvents.

J Biotechnol, 1994 May 15, 34(2), 133 - 47
Cell cycle dynamics of microcarrier cultures; Hawboldt KA et al.; In anchorage-dependent cell microcarrier cultures knowledge of the cell's growth kinetics is necessary in order to design and successfully operate bioreactors, particularly on a large scale . However, in addition to growth kinetics, an understanding of the physiological state of the culture is also important . In this paper the cell cycle progression of Vero and MRC-5 microcarrier cultures have been observed utilizing a flow cytometer . Flow cytometry analysis enabled the differentiation of the various phases of the cell cycle as the culture moved from initial inoculation to the stationary, or confluent stage . Not only was the flow cytometer able to distinguish contact inhibited cells from noncontact inhibited cells, but the measured fraction of contact inhibition cells were found to be in agreement with fractions predicted from a previously developed cellular automation model for microcarrier cultures . Further, the data from the stationary phase was used to quantify the death rate in microcarrier cultures.

Ann N Y Acad Sci, 1994 May 2, 721, 218 - 33
The porcine mammary gland as a bioreactor for complex proteins; Morcol T et al.; The similar biological activity of rhPC and hPC indicates that porcine mammary gland can perform many of the processing reactions necessary for recombinant synthesis of complex human proteins and produce them at levels suitable for industrial bioreactor applications . The health of the transgenic pigs appeared unaffected by the expression of high levels of the heterologous protein . We suggest that one of the advantages of using the mammary gland as a bioreactor appears to be the high cell density relative to that of cell culture.

Ann N Y Acad Sci, 1994 May 2, 721, 194 - 207
Analysis of foreign protein overproduction in recombinant CHO cells . Effect of growth kinetics and cell cycle traverse; Gu MB et al.; Intracellular foreign protein (beta-galactosidase) expression in recombinant CHO cell lines in continuous culture was analyzed by developing a mathematical model that includes the effects of metabolic burden and cell cycle dependence of intracellular foreign protein expression . This combined growth kinetic and cell cycle model, assuming S- or G1-phase-dependent expression, was stimulated to predict productivity on a single-cell and culture-volume basis in continuous cultures . In the case of S-phase-dependent expression, the intracellular foreign protein level increases monotonically, but in the case of G1-phase-dependent expression it decreases monotonically with increasing dilution rate . Also, the trends of foreign protein concentration in the culture volume differ significantly between S- and G1-dependent expression kinetics . Thus, the cell cycle dependency of foreign protein expression should be included in process optimization concepts and operating strategies of continuous bioreactors.

Artif Organs, 1994 May, 18(5), 371 - 84
Development of a bioartificial liver using isolated hepatocytes; Dixit V; Severe liver disease is very often life-threatening and dramatically diminishes quality of life . Liver support systems based on detoxification alone have been proved ineffective because they cannot correct biochemical disorders . An effective artificial liver support system should be capable of carrying out the liver's essential processes, such as synthetic and metabolic functions, detoxification, and excretion . It should be capable of sustaining patients with fulminant hepatic failure, preparing patients for liver transplantation when a donor liver is not readily available (i.e., bridge to transplantation), and improving the survival and quality of life for patients for whom transplantation is not a therapeutic option . Recent advances in cell biology, tissue culture techniques, and biotechnology have led the way for the potential use of isolated hepatocytes in treating an array of liver disorders . Isolated hepatocytes may be transplanted to replace liver-specific deficiencies or as an important element of an auxiliary hybrid, bioartificial extracorporeal liver support device, which are important therapeutic applications for treating severe liver disease . Recently, several hepatocyte-based liver support systems have been proposed . Although there is no current consensus on its eventual design configuration, the hollow fiber hepatocyte bioreactor shows the greatest promise . Furthermore, application of tissue engineering technology, based on cell-surface interaction studies proposed by our group and others, has enhanced interest in the development of highly efficient hybrid, bioartificial, liver support devices.

Aviat Space Environ Med, 1994 May, 65(5 Suppl), A131 - 7
Environmental biotechnology research: an overview; Spain JC; Cleanup and treatment of hazardous wastes incur major operational costs for the U.S . Air Force . Bioremediation can provide a cost-effective alternative to traditional technologies for a wide range of natural organic compounds such as jet fuel . Bioventing and natural attenuation are emerging as treatments of choice in many instances . Synthetic organic chemicals are much more resistant to biodegradation . However, recent advances in biotechnology allow the development of strains able to use nitro- and chloro-substituted organic compounds as their sole source of carbon and energy . Current basic research is focused on expanding the range of synthetic chemicals amenable to biodegradation . At the same time, development of appropriate bioreactors and models for scale up are essential for practical application of the technology.

Anal Chem, 1994 May 1, 66(9), 1485 - 9
Continuous-flow/stopped-flow system incorporating two rotating bioreactors in tandem: application to the determination of alkaline phosphatase activity in serum; Raba J et al.; Two rotating bioreactors in tandem have been incorporated into a continuous-flow/stopped-flow sample/reagent processing setup for the determination of alkaline phosphatase (EC3.1.3.1) activity in serum samples . The strategy circumvents incompatibility of buffer systems as well as that of the immobilized enzymes utilized in the bioreactors (alkaline phosphatase and alcohol oxidase, EC 1.1.3.13) . The determination is indirect in nature although recorded responses are directly related to the enzyme activity in the sample . It couples the following enzyme-catalyzed reactions: (1) hydrolysis of p-nitrophenyl dihydrogen phosphate catalyzed by alkaline phosphatase, (2) enzymatic reaction between unreacted p-nitrophenyl dihydrogen phosphate with methanol, and (3) conversion of the residual methanol to the corresponding aldehyde and H2O2, catalyzed by alcohol oxidase . The H2O2 is amperometrically determined at a stationary Pt-ring electrode (applied potential + 0.600 V vs a Ag/AgCl, 3.0 M NaCl reference).

FEMS Microbiol Rev, 1994 May, 14(1), 69 - 74
Continuous ethanol production by flocculating yeast in the fluidized bed bioreactor; Wieczorek A et al.; Continuous fermentation by a highly flocculant strain of the yeast Saccharomyces cerevisiae was carried out in a tower fluidized-bed bioreactor . The synthetic and molasses media with a total sugar concentration of 17% (w/v) were used for fermentation . Different dilution rates were tested . Stable cell densities of 50 kg m-3 (dry weight) were maintained for all dilution rates . The ethanol productivity was increasing linearly with dilution rates up to 15-20 kg m-3 h-1 . Aeration of the culture stabilized flocculating activity and viability of yeast and also permitted long-term operation of the bioreactor.

Biotechnol Prog, 1994 May-Jun, 10(3), 340 - 3
Degradation of pesticides in a continuous-flow two-phase microemulsion reactor; Komives C et al.; Biocatalytic processes in reversed micelles are limited by the need for continuous reactors in which reaction and separation are feasible . In this paper, we report the design and testing of a continuous-flow centrifugal bioreactor for the enzyme-catalyzed hydrolysis of the common pesticide paraoxon in reverse micelles of Tween 85 . In this system, organophosphorus hydrolase catalyzes hydrolysis of the pesticide in an organic-organic two-phase system . The enzyme is retained in one of the phases by encapsulation in reversed micelles, while the other phase is under a continuous flow . This liquid-liquid plug-flow reactor does not leach enzyme because the enzyme is not soluble in the flowing organic phase that contains the paraoxon.

Biotechnol Prog, 1994 May-Jun, 10(3), 277 - 83
Sucrose conversion by immobilized invertase in a multiple air-lift loop bioreactor; Bakker WA et al.; A new bioreactor series within one vessel, the multiple air-lift loop reactor (MAL), is introduced . In the MAL, a series of air-lift loop reactors is incorporated into one vessel . From residence time distribution studies, it was shown that the three-compartment MAL behaves as a series of three ideal mixers . A continuously operated MAL, containing immobilized invertase as a model biocatalyst, was evaluated . The advantage of approaching plug flow by using a bioreactor cascade could be shown by comparing substrate conversion in the three-compartment MAL to that in a single vessel at the same overall dilution rate . This was done for two sets of experimental conditions, which were chosen by using a previously developed model . Intrinsic kinetic parameters of the immobilized enzyme, needed for the model calculations, were determined experimentally . Model calculations gave good approximations of the results . The model incorporates external mass-transfer resistance and diffusion and reaction in the biocatalyst beads.

Biochem Pharmacol, 1994 Apr 20, 47(8), 1357 - 63
Stereoselective S-oxidation of flosequinan sulfide by rat hepatic flavin-containing monooxygenase 1A1 expressed in yeast; Kashiyama E et al.; Rat hepatic flavin-containing monooxygenase (FMO) 1A1 expressed in yeast catalysed the S-oxidation of flosequinan sulfide (7-fluoro-1-methyl-3-methylthio-4-quinolone) to R(+)-flosequinan (sulfoxide form, R(+)-7-fluoro-1-methyl-3-methylsulfinyl-4-quinolone) but not to S(-)-flosequinan, and did not catalyse the oxidation of R(+)- and S(-)-flosequinan to flosequinan sulfone . The Km and Vmax for the stereoselective S-oxidation were 33 microM and 6.2 nmol per min per mg of microsomal protein, respectively . The S-oxidation was inhibited by 1-(1-naphthyl)-2-thiourea and thiobenzamide . n-Octylamine activated the S-oxidation with little change in stereoselectivity . The ability of the recombinant yeast to produce R(+)-flosequinan from flosequinan sulfide could be maintained for at least 2 days and exemplifies the value of a recombinant yeast expressing FMO as a stereoselective bioreactor.

J Biotechnol, 1994 Apr 15, 33(3), 249 - 58
Optimization of the physiochemical parameters for the culture of Bombyx mori insect cells used in recombinant protein production; Zhang J et al.; Silkworm (Bombyx mori, Bm5) cell growth at different environmental conditions was systematically studied in controlled bioreactors . The physiochemical parameters, which affect the growth of Bm5 cells, were defined and optimized . The growth rate was maximal at a medium osmolality of about 370 mosm kg-1; and more than 90% of the maximum growth rate was achieved with osmolality between 350 and 385 mosm kg-1 . The optimum pH for Bm5 cell growth was from 6.10 to 6.30 . Moreover, the effect of dissolved oxygen concentrations (higher than 20% air saturation) on growth was minimal . However, the maximum cell density decreased markedly with dissolved oxygen tensions less than 20% . The shear protection additive Pluronic F-68 had a very important effect on the growth of Bm5 cells in bioreactors with an optimum concentration of 0.2% (w/v) . Compared to the original culture condition, the optimized conditions resulted in a twofold increase in cell yield and a reduced population doubling time from 48 to 36 h . Furthermore, the yield of a recombinant protein (CAT) was increased substantially in the optimized cultures.

Hepatology, 1994 Apr, 19(4), 1023 - 8
A new three-dimensional culture system for hepatocytes using reticulated polyurethane; Sato Y et al.; Poly-N-para-vinylbenzyl-lactonamide (PVLA)-coated reticulated polyurethane (PVLA-RPU) has been employed for the long-term maintenance of primary rat hepatocyte cultures . After 3 days of incubation of 2 x 10(7) hepatocytes/cm3 embedded in PVLA-RPU discs and kept in culture medium, most cells showed typical hepatocyte morphology, with some bile canaliculus-like intercellular spaces among the hepatocytes on examination with scanning and transmission electron microscopy . The cells were attached to the surface of the PVLA-RPU and formed multicellular spheroids in the reticulated pores . The hepatocytes maintained various liver-specific functions such as albumin secretion, ammonium metabolism, urea synthesis and gluconeogenesis, and they were viable . The liver-specific functions could be maintained for more than 1 month when the cells were kept in the rats' peritoneal cavities . This new system may be useful as a bioreactor for an artificial liver.

Berl Munch Tierarztl Wochenschr, 1994 Apr, 107(4), 123 - 7
{Continuous production of FMD virus in a two-step bioreactor system}; Roth F et al.; The so-called Goettingen two step bioreactor system has been tested in a pilot-project in Argentina to produce FMD-virus . With a 10-1-reactor a cell density of 3.5-4 x 10(6) cells/ml could be maintained and virus titres of up to 10(7.2) TCID/ml were reached . A correlation existed between inoculum titre and incubation period of virus . With lower inoculum a higher titre of harvested virus and a shorter incubation period were obtained . In spite of considerable problems with peripheral infrastructure and laboratory equipment the bioreactor system did work reliably . The project was terminated before final results of virus yield could be reached.

Nat Biotechnol, 1999 Nov, 17(11), 1087 - 90
Seminal vesicle production and secretion of growth hormone into seminal fluid; Dyck MK et al.; Production of foreign proteins in the tissues of transgenic animals represents an efficient and economical method of producing therapeutic and pharmaceutical proteins . In this study, we demonstrate that the mouse P12 gene promoter specific to the male accessory sex gland can be used to generate transgenic mice that express human growth hormone (hGH) in their seminal vesicle epithelium . The hGH is secreted into the ejaculated seminal fluids with the seminal vesicle lumen contents containing concentrations of up to 0.5 mg/ml . As semen is a body fluid that can be collected easily on a continuous basis, the production of transgenic animals expressing pharmaceutical proteins into their seminal fluid could prove to be a viable alternative to use of the mammary gland as a bioreactor.

In Vitro Cell Dev Biol Anim, 1999 Oct, 35(9), 501 - 9
Induction of three-dimensional assembly of human liver cells by simulated microgravity; Khaoustov VI et al.; The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable . Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types . The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity . PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS . PHLC aggregates were readily formed and increased up to 1 cm long . The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds . While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long . Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts . Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions . Albumin mRNA was expressed throughout the 60-d culture. , , , , . A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes . This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.

Ann Biomed Eng, 1999 Sep-Oct, 27(5), 656 - 62
Method for quantitative analysis of glycosaminoglycan distribution in cultured natural and engineered cartilage; Martin I et al.; Cartilage tissue engineering can provide a valuable tool for controlled studies of tissue development . As an example, analysis of the spatial distribution of glycosaminoglycans (GAG) in sections of cartilaginous tissues engineered under different culture conditions could be used to correlate the effects of environmental factors with the structure of the regenerated tissue . In this paper we describe a computer-based technique for quantitative analysis of safranin-O stained histological sections, using low magnification light microscopy images . We identified a parameter to quantify the intensity of red color in the sections, which in turn was proportional to the biochemically determined wet weight fraction of GAG in corresponding tissue samples, and to describe the spatial distribution of GAG as a function of depth from the section edge . A broken line regression model was then used to determine the thickness of an external region, with lower GAG fractions, and the spatial rate of change in GAG content . The method was applied to the quantitation of GAG distribution in samples of natural and engineered cartilage, cultured for 6 weeks in three different vessels: static flasks, mixed flasks, and rotating bioreactors.

Clin Orthop, 1999 Oct, (367 Suppl), S254 - 72
Biomechanical factors in tissue engineered meniscal repair; Setton LA et al.; Damage to the meniscus after trauma or injury is associated with detrimental changes in joint function that can lead to pain, disability, and degenerative joint changes . Recently, tissue engineering strategies for meniscal repair have been suggested including using biocompatible grafts as a substrate for regeneration, and cellular supplementation to promote remodeling and healing . Little is known, however, about the contributions of these novel repair strategies to restoration of normal meniscal function . Biomechanical factors play a role in the design and synthesis of tissue engineered biomaterials and bioreactors, and also are important for evaluating the efficacy of these new strategies for restoring normal meniscal function . In this report, an overview is presented of biomechanical factors that are critical to meniscal function followed by a review of biomechanical considerations for the design and evaluation of tissue engineered strategies for meniscal repair . Recommendations for future study of biomechanical factors in tissue engineered meniscal repair also are provided.

Clin Orthop, 1999 Oct, (367 Suppl), S244 - 53
Building a meniscus . Biologic considerations; Arnoczky SP; Creating a tissue engineered meniscus requires that specific biologic considerations such as cell type, matrix scaffold, bioreactor design (in vitro, in vivo, or both), and environmental conditions (media, cytokines, physical stimuli) be addressed . Meniscal cells, fibroblasts, and mesenchymal stem cells have been proposed as potential cell sources and have been grown (either in vivo or in vitro) on various cell matrices including collagen based scaffolds, biodegradable polymers, and small intestine submucosa . In addition, various environmental factors (nutrients, growth factors) have been used to optimize cell proliferation in vitro . Although these tissue engineering paradigms have produced a meniscuslike construct, the long term functional capabilities of this engineered tissue have yet to be proven . The concept of tissue engineering holds great promise for the generation of tissues or organs unable to repair or regenerate themselves . In terms of the meniscal tissue, however, the principles of tissue engineering may be best used to enhance and optimize the natural regeneration of the meniscus.

Clin Orthop, 1999 Oct, (367 Suppl), S46 - 58
Frontiers in tissue engineering . In vitro modulation of chondrogenesis; Freed LE et al.; Cartilage tissue engineering can provide functional cartilaginous constructs that can be used for controlled in vitro studies of chondrogenesis and potentially for in vivo articular cartilage repair . Ideally, engineered cartilage should be indistinguishable from native articular cartilage with respect to zonal organization, biochemical composition, and mechanical properties . In the model system presented here, chondrogenic cells are expanded in vitro as required, seeded onto three-dimensional polymeric scaffolds, and cultured in bioreactor vessels . During the course of in vitro cultivation, construct cellularity plateaus at a physiologic level, fractions of glycosaminoglycan and Type II collagen increase progressively, and the scaffold biodegrades . Construct structure (composition, morphology) and function (biosynthetic activity, mechanical properties) depend on cultivation conditions . This paper reviews recent studies of in vitro modulation of chondrogenesis by: (1) cell seeding density and source; (2) the tissue regeneration template; (3) biochemical regulatory signals; (4) mixing, mass transport and hydrodynamic forces; and (5) cultivation time . Key requirements and some of the critical research needs for successful cartilage tissue engineering are discussed.

Int J Artif Organs, 1999 Aug, 22(8), 566 - 72
A simple method for hepatocyte attachment in hollow fibre bioreactors; Karlik W et al.; A new method for hepatocyte attachment in hollow fibre (HF) bioreactors was proposed and verified . A flow of medium with suspended hepatocytes, evoked by transmembrane pressure (TMP), and directed across the membrane into the fibre lumen, has accelerated and improved hepatocyte contact with the HF . It was found that seeding of hepatocytes onto the membrane was optimal at TMP of 50-80 mmHg . Ammonia utilisation and ureagenesis rates in hepatocytes seeded in the bioreactor suggests that the proposed method warrants proper conditions for cell functionality and allows for extended culture of hepatocytes in HF bioreactors . It is speculated that time cutback between introduction of hepatocytes into the bioreactor and the start of the cell attachment process, accomplished by the presented method, leads to substantially improved recovery of freshly isolated hepatocytes, and consequently to better overall performance of HF bioreactor.

Appl Biochem Biotechnol, 1999 Jul, 81(1), 1 - 11
Fungal morphology in submerged cultures and its relation to glucose oxidase excretion by recombinant Aspergillus niger; el-Enshasy H et al.; The effect of culture conditions such as medium composition and shear stress on the fungal pellet morphology in shake-flask cultures and its relation to glucose oxidase (GOD) excretion by recombinant Aspergillus niger NRRL 3 (GOD 3-18) was investigated . It was shown that culture conditions resulting in the formation of smaller fungal pellets with an increased mycelial density result in higher yields of exocellular GOD . The pellets obtained in shake-flask cultures showed distinct layers of mycelial density with only the thin outer layer consisting of a dense mycelial network . The performance of the recombinant strain and the process of pellet formation was also analyzed during batch cultivation in a stirred-tank bioreactor . It was shown that the process of pellet formation occurred in two steps: (1) aggregation of free spores to spore clusters with subsequent germination and formation of small aggregates surrounded by a loose hyphal network, and (2) aggregation of the primary aggregates to the final full-size pellets . The fungal pellets formed during bioreactor cultivation were smaller, did not show large differences in mycelial density, and were more efficient with respect to the production of exocellular GOD . The decreasing pellet size also correlated with an increased mycelial density, indicating an improvement of the transport of nutrients to the inner parts of the pellet.

Appl Microbiol Biotechnol, 1999 Sep, 52(3), 360 - 5
Benzene/toluene/p-xylene degradation . Part II . Effect of substrate interactions and feeding strategies in toluene/benzene and toluene/p-xylene fermentations in a partitioning bioreactor; Collins LD et al.; A two-phase aqueous/organic partitioning bioreactor scheme was used to degrade mixtures of toluene and benzene, and toluene and p-xylene, using simultaneous and sequential feeding strategies . The aqueous phase of the partitioning bioreactor contained Pseudomonas sp . ATCC 55595, an organism able to degrade benzene, toluene and p-xylene simultaneously . An industrial grade of oleyl alcohol served as the organic phase . In each experiment, the organic phase of the bioreactor was loaded with 10.15 g toluene, and either 2.0 g benzene or 2.1 g p-xylene . The resulting aqueous phase concentrations were 50 mg/l, 25 mg/l and 8 mg/l toluene, benzene and p-xylene respectively . The simultaneous fermentation of benzene and toluene consumed these compounds at volumetric rates of 0.024 g l-1 h-1 and 0.067 g l-1 h-1, respectively . The simultaneous fermentation of toluene and p-xylene consumed these xenobiotics at volumetric rates of 0.066 g l-1 h-1 and 0.018 g l-1 h-1, respectively . A sequential feeding strategy was employed in which toluene was added initially, but the benzene or p-xylene aliquot was added only after the cells had consumed half of the initial toluene concentration . This strategy was shown to improve overall degradation rates, and to reduce the stress on the microorganisms . In the sequential fermentation of benzene and toluene, the volumetric degradation rates were 0.056 g l-1 h-1 and 0.079 g l-1 h-1, respectively . In the toluene/p-xylene sequential fermentation, the initial toluene load was consumed before the p-xylene aliquot was consumed . After 12 h in which no p-xylene degradation was observed, a 4.0-g toluene aliquot was added, and p-xylene degradation resumed . Excluding that 12-h period, the microbes consumed toluene and p-xylene at volumetric rates of 0.074 g l-1 h-1 and 0.025 g l-1 h-1, respectively . Oxygen limitation occurred in all fermentations during the rapid growth phase.

Appl Microbiol Biotechnol, 1999 Sep, 52(3), 354 - 9
Benzene/toluene/p-xylene degradation . Part I . Solvent selection and toluene degradation in a two-phase partitioning bioreactor; Collins LD et al.; A two-phase organic/aqueous reactor configuration was developed for use in the biodegradation of benzene, toluene and p-xylene, and tested with toluene . An immiscible organic phase was systematically selected on the basis of predicted and experimentally determined properties, such as high boiling points, low solubilities in the aqueous phase, good phase stability, biocompatibility, and good predicted partition coefficients for benzene, toluene and p-xylene . An industrial grade of oleyl alcohol was ultimately selected for use in the two-phase partitioning bioreactor . In order to examine the behavior of the system, a single-component fermentation of toluene was conducted with Pseudomonas sp . ATCC 55595 . A 0.5-1 sample of Adol 85 NF was loaded with 10.4 g toluene, which partitioned into the cell containing 1 l aqueous medium at a concentration of approximately 50 mg/l . In consuming the toluene to completion, the organisms were able to achieve a volumetric degradation rate of 0.115 g l-1 h-1 . This system is self-regulating with respect to toluene delivery to the aqueous phase, and requires only feedback control of temperature and pH.

Can J Microbiol, 1999 Aug, 45(8), 653 - 7
Enhanced benzaldehyde formation by a monokaryotic strain of Pycnoporus cinnabarinus using a selective solid adsorbent in the culture medium; Lomascolo A et al.; A monokaryotic strain of the white-rot fungus Pycnoporus cinnabarinus was shown to produce, in a 2-L bioreactor culture, 100 mg.L-1 benzaldehyde (bitter almond aroma) from L-phenylalanine with a productivity of 33 mg.L-1.day-1 . The addition of HP20 resin, a styrene divinylbenzene copolymer highly selective for benzaldehyde, enabled an eightfold increase in the production of benzaldehyde and a twofold increase in productivity . In the presence of HP20 resin, the production of 790 mg.L-1 benzaldehyde was concomitant with the synthesis of cinnamic acid derivatives of high organoleptic notes such as cinnamaldehyde, cinnamyl alcohol, and methyl cinnamate.

Mar Biotechnol (NY), 1999 Sep, 1(5), 495 - 0507
Bioprocess Intensification for Production of Novel Marine Bacterial Antibiotics Through Bioreactor Operation and Design; Marwick JD et al.; : There is a lack of research into bioreactor engineering and fermentation protocol design in the field of marine bacterial antibiotic production . Most production strategies are carried out at the shake-flask level and lack a mechanistic understanding of the antibiotic production process, offering poor prospects for successful scale-up . This review shows that data need to be collated on media and physical optima differences between the trophophase and idiophase, along with investigations into the control mechanisms for biosynthesis, to allow implementation of novel fermentation protocols . Immobilization may play a part in bioprocess intensification of marine bacterial antibiotic production, through again this area is understudied . Similarly, mass transfer and shear stress data of fermentations are needed to provide the bioreactor design requirements to intensify antibiotic biosynthesis, with process scale-up in mind . The application of bioprocess intensification methods to the production of antibiotics (and other metabolites) from marine microbes will become an important strategy for improving supply of natural products, in order to assess their suitability as chemotherapeutic drugs.

Biotechnol Bioeng, 1999 Dec 5, 65(5), 589 - 99
Extended serial passaging of mammalian neural stem cells in suspension bioreactors; Kallos MS et al.; Neural stem cells (NSCs) are primitive cells that are the "parent" cells of all the cells in the central nervous system (CNS) . Their discovery in 1992 opened the door to a multitude of potential therapies and treatments to cure neurodegenerative diseases such as Parkinson's disease, multiple sclerosis, and Huntington's disease, which affect millions of people worldwide and cost billions of dollars in health care each year . This study proposes optimal serial passaging protocols so that mammalian neural stem cells can effectively be grown in suspension culture . We examined stationary culture passaging protocols and developed our own optimal procedure . Also examined was the effect of serially cultivating the neural stem cells in suspension culture for an extended period of time . The cells were grown for over 35 days in suspension with an overall multiplication ratio of over 10(7) with no decrease in growth rate, maximum cell density, or viability . The cells also remained karyotypically normal through 25 doublings and retained their ability to be differentiated into all the major cell types of the CNS-neurons, astrocytes, and oligodendrocytes . For the first time, mammalian neural stem cells were grown on a larger scale in suspension culture and maintained their stem cell characteristics . A semicontinuous scheme for large-scale production is also presented .

Biotechnol Bioeng, 1999 Dec 5, 65(5), 558 - 71
Mass balance modeling of vanillin production from vanillic acid by cultures of the fungus Pycnoporus cinnabarinus in bioreactors; Bernard O et al.; A systematic two-step procedure for the structural identification of bioprocesses is followed in order to establish a mechanistic model for vanillin production by Pycnoporus cinnabarinus . The first step is devoted to the identification of the underlying reaction structure and the development of a validated mass balance model for the growth of P . cinnabarinus and the biotransformation of vanillic acid into vanillin . The second step is devoted to the kinetic modeling, namely, the estimation of the reaction rates and the calibration of the kinetic parameters . The whole procedure leads to the final set up of a simulation model of the process . The results are supported by the data from five cultures of P . cinnabarinus in bioreactors .

Biotechnol Bioeng, 1999 Dec 5, 65(5), 523 - 8
Conditionally adherent growth of serum-independent CHO cells for automated drug screening and biopharmaceutical production; Gandor CR et al.; SSF3 is a CHO cell line adapted for growth in protein-free medium . It grows in suspension unless serum-derived attachment factors such as vitronectin are added to the medium . Serum-independent cell lines, which adhere to the substrate after induction with dexamethasone or constitutively, were created by transfection with a human vitronectin gene under control of the mouse mammary tumor-virus promoter . Substrate attachment and SSF3VN-cell spreading could be prevented with an RGD peptide (arginine-glycine-aspartic acid) confirming that attachment is mediated by an intregrin receptor . Hormone-inducible attachment could be blocked by glucocorticoid antagonist promegestone . All steps in the isolation of stable transfected SSF3VN cell lines could be done in a chemically defined medium avoiding the risk of introduction of serum-derived infectious agents . SSF3VN cells could be grown in protein-free medium in solid-phase large-scale bioreactors . Application in microplates as used in high-throughput screening was demonstrated in an assay of Ca(2+) release from internal stores induced by agonist-binding to recombinant human metabotropic glutamate receptor hmGluR1b .

Biotechnol Prog, 1999 Oct 1, 15(5), 834 - 844
Reactor Design Issues for Synthesis-Gas Fermentations; Bredwell MD et al.; Synthesis gas is readily obtained by gasifying coal, oil, biomass, or waste organics and represents an abundant, potentially inexpensive, feedstock for bioprocessing . The primary components of synthesis gas, carbon monoxide and hydrogen, can be converted into methane, organic acids, and alcohols via anaerobic fermentations . Bioconversion of synthesis gas is an attractive alternative to catalytic processing because the biological catalysts are highly specific and often more tolerant of sulfur contaminants than inorganic catalysts . However, because the aqueous solubilities of carbon monoxide and hydrogen are low, synthesis-gas fermentations are typically limited by the rate of gas-to-liquid mass transfer . Consequently, a major engineering challenge in commercial development of synthesis-gas fermentations is to provide sufficient gas mass transfer in an energy-efficient manner . This paper reviews recent progress in the development of synthesis-gas fermentations, with emphasis on efforts to increase the efficiency of gas mass transfer . Metabolic properties of several microbes able to ferment synthesis gas are described . Results of synthesis-gas fermentations conducted in various bioreactor configurations are summarized . Recent results showing enhancement of synthesis-gas fermentations using microbubble dispersions are presented, and studies of the mass-transfer and coalescence properties of microbubbles are described.

Biotechnol Appl Biochem, 1999 Oct, 30 ( Pt 2), 109 - 12
Towards molecular farming in the future: using plant-cell-suspension cultures as bioreactors; Fischer R et al.; Plant-suspension cells are an in vitro system that can be used for recombinant protein production under carefully controlled certified conditions . Plant-suspension cells can be grown in shake flasks or fermenters to produce secondary metabolites, like vincristine and vinblastine, and to produce recombinant proteins after transformation . This review article focuses on discussing the generation of transformed suspension-cell lines expressing recombinant proteins, like antibodies, and recombinant-protein downstream processing and purification.

Biotechnol Appl Biochem, 1999 Oct, 30 ( Pt 2), 101 - 8
Towards molecular farming in the future: moving from diagnostic protein and antibody production in microbes to plants; Fischer R et al.; Molecular farming of pharmaceuticals in plants has the potential to provide almost unlimited amounts of recombinant proteins for use in disease diagnosis and therapy . Transgenic plants are attracting interest as bioreactors for the inexpensive production of large amounts of safe, functional, recombinant macromolecules, such as blood substitutes, vaccines and antibodies . In some cases, the function of expressed recombinant proteins can be rapidly analysed by expression in microbes or by transient expression in intact or virally infected plants . Protein production can be increased by upscaling production in fermenters, using yeast- or plant-suspension cells or by using transient-expression systems . Stable transgenic plants can be used to produce leaves or seeds rich in the recombinant protein for long-term storage or direct processing. , , , , . This demonstrates the promise for using plants as bioreactors for the molecular farming of recombinant therapeutics, diagnostics, blood substitutes and antibodies . We anticipate that this technology has the potential to greatly benefit human health by making safe recombinant pharmaceuticals widely available.

J Ind Microbiol Biotechnol, 1999 Aug, 23(2), 133 - 137
Dynamics and stability analysis of the growth and astaxanthin production system of Haematococcus pluvialis; Zhang XW et al.; This paper investigated high cell density cultivation of Haematococcus pluvialis for astaxanthin production in 3.7-L bioreactors . A biomass concentration of 2.74 g L-1and an astaxanthin yield of 64.4 mg L-1 were obtained . Based on the experimental results, a new and simple dynamic model is proposed, differing from Monod kinetics, to describe cell growth, product formation and substrate consumption . Good agreement was found between the model predictions and experimental data . The model revealed that there was cell growth inhibition on product formation and product feedback compensation for substrate consumption, but no substrate inhibition or product inhibition of cell growth . Stability analysis demonstrated that no multiplicity of steady states was observed; the unique positive steady state was locally asymptotically stable; and the effect of dilution rate on steady states was greater than that of the initial substrate concentration.

Biotechnol Bioeng, 1999 Nov 20, 65(4), 468 - 73
Design and scale up of a process for manganese peroxidase production using the hypersecretory strain Phanerochaete chrysosporium I-1512; Herpoel I et al.; Manganese peroxidase (MnP) production was performed in a airlift bioreactor in which Phanerochaete chrysosporium I-1512, an MnP hypersecretory strain, was immobilized on a stainless steel mesh . Production was scaled up from a 2.5-L bench scale to a 100-L bioreactor . The yield of MnP was increased 2-fold and reached 6600 U L(-1) . These results indicate the feasibility of MnP production on a medium scale, which promises sufficient MnP availability for its use in pulp bleaching at industrial scale .

Chin J Biotechnol, 1998, 14(3), 187 - 93
Optimization in hybridoma cell culture; Zhang Y et al.; Based on the formation kinetics of monoclonal antibody, the bench-scale culture of hybridoma cells in a bioreactor was optimized by the application of perfusion culture mode, the supplement of potassium acetate, and the fortification of nutrients . When the bioreactor was operated with the daily perfusion of medium of 1/2 working volume, viable cell density in bioreactor was 11 x 10(5) cells/ml and antibody concentration 28 mg/L . After supplement with 1 g/L potassium acetate, the cell density kept constant and antibody production was improved to 38 mg/L . The fortification of nutrients such as amino acids and vitamins raised the cell density up to 42 x 10(5) cells/ml and, responsively, antibody concentration to 94 mg/L.

J Microbiol Immunol Infect, 1998 Sep, 31(3), 146 - 50
Optimized conditions for growth and fermentation of Helicobacter pylori; Wang JT et al.; This study attempted to optimize the growth conditions of Helicobacter pylori by comparing the growth of H . pylori in an anaerobic jar and an incubator . It was found that the primary isolation rate of H . pylori from gastric biopsies was higher in an anaerobic incubator (28/30) than in an anaerobic jar (21/30) . In addition, growth rate in the subsequent passage was also higher in an incubator . By comparing different supplements in the liquid media, it was found that most strains grew best in Brucella broth containing 5% fetal calf serum, and some strains grew well with the supplement of 0.2% of beta-cyclodextrin . A large scale culture using a bioreactor indicated that a defoaming agent, polypropylenglycol, profoundly inhibited the growth of H . pylori . However, fermentation could be successfully performed by continuously infusing mixed airs composed of 5% O2, 10% CO2 and 85% N2 . Moreover, the production of secreted proteins of H . pylori strains grown in the bioreactor was the same as that in an anaerobic jar . Results presented herein can be used to further optimize conditions for enhancing the growth of H . pylori.

Dev Biol Stand, 1999, 98, 23 - 37; discussion 73-4
Production of influenza virus in serum-free mammalian cell cultures; Merten OW et al.; Human influenza viruses are routinely isolated and grown in a variety of mammalian cell substrates . However, influenza viruses for use as inactivated vaccine are still produced in embryonated eggs . Using a perfusion culture-based bioreactor process using serum-free medium, both human and equine influenza viruses of different types and subtypes could be produced to high titres . Classical DEAE-dextran microcarriers were found to be more suitable than polyester sponge carriers for virus production . In addition, MDCK cells grown in serum-free medium were further validated as the most suitable cell substrate compared to Vero and BHK-21 C13 cells for large scale virus production of influenza virus . Finally, to minimize potential contamination by adventitious agents, it was demonstrated that a new serum-free medium in which all animal-derived products are replaced by a plant extract, efficiently supports the growth of MDCK cells as well as the production of influenza virus in the presence of trypsin when using the perfusion bioreactor process.

Biol Chem, 1999 Jul-Aug, 380(7-8), 825 - 39
Molecular farming of recombinant antibodies in plants; Fischer R et al.; 'Molecular farming' is the production of recombinant proteins in plants . It is intended to harness the power of agriculture to cultivate and harvest transgenic plants producing recombinant therapeutics . Molecular farming has the potential to provide virtually unlimited quantities of recombinant antibodies for use as diagnostic and therapeutic tools in both health care and the life sciences . Importantly, recombinant antibody expression can be used to modify the inherent properties of plants, for example by using expressed antipathogen antibodies to increase disease resistance . Plant transformation is technically straightforward for model plant species and some cereals, and the functional expression of recombinant proteins can be rapidly analyzed using transient expression systems in intact or virally infected plants . Protein production can then be increased using plant suspension cell production in fermenters, or by the propagation of stably transformed plant lines in the field . Transgenic plants can be exploited to produce organs rich in a recombinant protein for its long-term storage . This demonstrates the promise of using transgenic plants as bioreactors for the 'molecular farming' of recombinant therapeutics, blood substitutes and diagnostics, such as recombinant antibodies.

Biodegradation, 1999 Jun, 10(3), 219 - 33
Two-liquid-phase bioreactors for enhanced degradation of hydrophobic/toxic compounds; Deziel E et al.; Two-liquid-phase culture systems involve the addition of a water-immiscible, biocompatible and non-biodegradable solvent to enhance a biocatalytic process . Two-liquid-phase bioreactors have been used since the mid-seventies for the microbial and enzymatic bioconversion of hydrophobic/toxic substrates into products of commercial interest . The increasing popularity of bioremediation technologies suggests a new area of application for this type of bioreactor . The toxicity and the limited bioavailability of many pollutants are important obstacles that must first be overcome in order to improve biodegradation processes . Two-liquid-phase bioreactors have the potential to resolve both limitations of biotreatment technologies by the enhancement of the mass-transfer rate of compounds with low bioavailability, and by the controlled delivery of apolar toxic compounds . This technology can also be useful in accelerating the enrichment of microorganisms degrading problematic pollutants . In this paper, we discuss the application of two-liquid-phase bioreactors to enhance the biodegradation of toxic/poorly bioavailable contaminants . Important microbial mechanisms involved in this type of system are described . Uptake of the substrates can be achieved by microorganisms freely dispersed in the aqueous phase and/or bound at the interface between the aqueous and the immiscible phases . Production of surface-active compounds and adhesion abilities are microbial features involved in the process . General guidelines for the design of two-liquid-phase bioreactors for biodegradation purposes are presented . Solvent selection should be established on specific criteria, which depend on the characteristics of target compound(s) and the microorganism(s) implicated in the biodegradation process . The central importance of maximizing the interfacial surface area is highlighted . The potential of this approach as an alternative to current biotreatment technologies is also discussed.

J Hepatol, 1999 Sep, 31(3), 542 - 9
Characterization of drug metabolizing activities in pig hepatocytes for use in bioartificial liver devices: comparison with other hepatic cellular models; Donato MT et al.; BACKGROUND/AIMS: The pig is considered the best donor of hepatocytes for bioartificial liver devices, but little is known about the metabolic capability of pig hepatocytes . Therefore, we have evaluated drug metabolizing activities in pig hepatocytes and liver microsomes and compared the results with those of man and other animal hepatic cellular models that are potential sources of cells for bioreactors, such as rat, rabbit and dog hepatocytes and hepatoma cell lines . METHODS: Total cytochrome P450 levels, six phase 1 activities representative of the most relevant cytochrome P450 enzymes (7-ethoxycoumarin O-deethylase, 7-ethoxy-, 7-methoxy- and 7-benzoxyresorufin O-dealkylases, coumarin 7-hydroxylase and p-nitrophenol hydroxylase), two phase 2 activities (glutathione S-transferase and UDP-glucuronyltransferase) and CYP-dependent regioselective testosterone metabolism were evaluated in in vitro models of different species . RESULTS: The pattern of specific cytochrome P450 activities and the metabolic profile of testosterone in intact hepatocytes were essentially the same as those measured in liver microsomes . Relatively low ethoxy-, methoxy-, and benzoxyresorufin O-dealkylation rates were found in pig liver microsomes and hepatocytes as compared to hepatic in vitro human models . However, in contrast with the other species studied, stereoselective testosterone oxidation profiles were practically identical in human and pig models . Finally, the metabolic capability of hepatoma cell lines was very limited in comparison with that of hepatocytes . CONCLUSIONS: Pig hepatocytes are able to maintain in culture the phase 1 and phase 2 activities found in liver microsomes . The high metabolic similarities found between pig and human hepatocytes lend support to the use of pig hepatocytes in bioartificial liver devices.

Appl Biochem Biotechnol, 1999 Jun, 80(3), 231 - 42
Alteration of glucose consumption kinetics with progression of baculovirus infection in Spodoptera frugiperda cells; Raghunand N et al.; We have used the initial-rate approach to characterize changes in the glucose consumption kinetics of baculovirus-infected Spodoptera frugiperda clone 9 (Sf9) cells with the progression of the infection process . The specific glucose consumption rate (qG) of cultured baculovirus-infected Sf9 cells was measured at 4, 8, 12, 16, and 24 h postinfection (h.p.i.) in media containing 4-35 mM glucose . Higher medium glucose concentrations resulted in higher final extracellular virus and recombinant beta-galactosidase yields . qG was related to the extracellular glucose concentration by means of a Michaelis-Menten relationship . The apparent Michaelis-Menten constant (K(m)) for glucose consumption was found not to change significantly during the progression of the infection process, and remained between 6.2 and 7.2 mM . However, the maximal specific glucose consumption rate (qGmax) was found to rapidly increase after infection, peaking at 16 h.p.i . at a value four times that for uninfected Sf9 cells . The kinetic analysis of glucose consumption rates in baculovirus-infected Sf9 cells presented here will aid in the optimal design and operation of bioreactor systems for the large-scale production of recombinant products from the baculovirus/insect cell system.

Biotechnol Bioeng, 1999 Nov 5, 65(3), 334 - 40
Effect of harvesting protocol on performance of a hollow fiber bioreactor; Gramer MJ et al.; In this study, a bioreactor subject to Starling flow in closed shell batch harvest mode was compared to two forms of additional forced extracapillary (EC) space convection including EC circulation and EC cycling . Despite the presence of Starling flow as the dominant EC convection mechanism in the batch harvest system, the bioreactor start up was fairly good . However, the antibody productivity of the batch harvest system fell off rapidly after day 20 resulting in only 4.5 g of antibody produced . EC circulation with flow parallel to the fibers had a slightly better start up than the batch harvest . However, the antibody productivity also dropped after day 20 with EC circulation, resulting in only 7.5 g of antibody produced . EC cycling with flow both parallel and perpendicular to the fibers resulted in a start up similar to that of EC circulation . However, in contrast to the other two systems, antibody productivity in the EC cycling system was stable over the 60-day experiment resulting in the production of 23 g of antibody . These results demonstrate the importance of inducing the proper flow distribution in the EC space to allow consistent and stable production in hollow fiber bioreactors .

Extremophiles, 1999 Aug, 3(3), 199 - 204
A microfiltration bioreactor to achieve high cell density in Sulfolobus solfataricus fermentation; Schiraldi C et al.; A novel technique is proposed to achieve higher cell yield in extremophile fermentation . Because the accumulation of toxic compounds is thought to be responsible for low biomass yields, a bioreactor has been designed based on a microfiltration hollow-fiber module located inside the traditional fermentation vessel . Using the cultivation of the thermoacidophilic archeon Sulfolobus solfataricus theta as a model, a biomass of 35gl(-1) dry weight was obtained which proved greater than that of 2gl(-1) obtained in batch fermentation . The bioreactor was characterized by running several fermentation experiments to check the high stability of the membrane module to sterilization cycles, high temperatures, and acidic pHs, even for prolonged periods of time . It was shown that the exhaust medium is unable to sustain growth for the presence of toxic compounds, and ultrafiltration and ion-exchange techniques were used in all the attempts to regenerate it . The results demonstrated the ability of the method to lower inhibitor concentrations and prolong the growth phase, thus achieving high cell density . Furthermore, they indicated that the toxic compounds are ionic species of less than 1kDa.

Planta Med, 1999 Aug, 65(6), 527 - 31
Biological properties of ulvan, a new source of green seaweed sulfated polysaccharides, on cultured normal and cancerous colonic epithelial cells; Kaeffer B et al.; Ulvans (from Ulva lactuca) constitute a dietary fiber structurally similar to the mammalian glycosaminoglycans but with unexplored biological or cytotoxic activities . From native low-viscosity preparations containing 33.5 molar % and 18.4 molar % of sulfate residues and uronic acid residues, respectively, we derived desulfated, reduced and desulfated-reduced polysaccharides with respectively 5.2, 2.9, and 4.5-4.9 molar % of sulfate residues and uronic acid residues . The effects of these preparations were examined on the adhesion, proliferation and differentiation of normal or tumoral colonic epithelial cells cultured in conventional (0.3-0.8 x 10(6) cells/ml) or rotating bioreactor (3-8 x 10(6) cells/ml) culture conditions . In conventional culture conditions, ulvan modified the adhesion phase and the proliferation of normal colonic cells and undifferentiated HT-29 cells according to their molecular weights and to the relative molar proportion of sulfate residues . From the native polysaccharides, we have screened sulfated ulvans (MW < 5,000) which inhibited the Caco-2 cell proliferation/differentiation program by inducing a low cell reactivity to Ulex europeaus-1 lectins in defined (p < 0.001) or serum-supplemented media (p < 0.01) but were inactive on normal colonocytes . In conclusion, this dietary fiber could be a source of oligosaccharides with a bioactivity, a cytotoxicity or a cytostaticity targeted to normal or cancerous epithelial cells.

J Biotechnol, 1999 May 28, 71(1-3), 105 - 31
Cybernetic model of the growth dynamics of Saccharomyces cerevisiae in batch and continuous cultures; Jones KD et al.; Growth of Saccharomyces cerevisiae on glucose in aerobic batch culture follows the well-documented diauxic pattern of completely fermenting glucose to ethanol during the first exponential growth phase, followed by an intermediate lag phase and a second exponential growth phase consuming ethanol . In continuous cultures over a range of intermediate dilution rates, the yeast bioreactor exhibits sustained oscillations in all the measured concentrations, such as cell mass, glucose, ethanol, and dissolved oxygen, the amounts of intracellular storage carbohydrates, such as glycogen and trehalose, the fraction of budded cells as well as the culture pH . We present here a structured, unsegregated model for the yeast growth dynamics developed from the 'cybernetic' modeling framework, to simulate the dynamic competition between all the available metabolic pathways . This cybernetic model accurately predicts all the key experimentally observed aspects: (i) in batch cultures, duration of the intermediate lag phase, sequential production and consumption of ethanol, and the dynamics of the gaseous exchange rates of oxygen and carbon dioxide; and (ii) in continuous cultures, the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitatin rate are changed.

J Biotechnol, 1999 May 28, 71(1-3), 7 - 16
Computing operating diagrams of bioreactors; Pavlou S; The operating diagram of a bioreactor is an illustrative way to present the effect that the operating conditions have on its long-term behavior . It can be constructed if a mathematical model of the bioreactor is available . The procedure for constructing the operating diagram consists in analyzing the dynamic behavior of the system of the differential equations of the model . Some methods are described that can be used in computing operating diagrams of bioreactors . They are based on numerical bifurcation techniques for systems of differential equations . Both cases of bioreactors with constant and periodically varying operating conditions are considered.

J Gastroenterol Hepatol, 1999 Aug, 14(8), 757 - 70
Extracorporeal support and hepatocyte transplantation in acute liver failure and cirrhosis; Riordan SM et al.; The relative shortage of donor organs and lack of immediate availability mean that many patients with acute liver failure die before orthotopic liver transplantation can be performed . An effective temporary liver support system could improve the chance of survival with or without a transplant being ultimately carried out . Recent technological advances resulting in improved maintenance of hepatocyte viability and function in culture and bioreactor designs which facilitate adequate perfusion of the cellular component and removal of products of cellular metabolism have led to the development of a number of bioartificial devices for liver support . Three such devices have undergone preliminary clinical evaluation in the setting of acute liver failure, with a statistically significant reduction in raised intracerebral pressure along with improvements in consciousness level and some biochemical parameters associated with treatment with one of these . Several other devices with different characteristics have shown promise in vitro and/or in animal models but await clinical evaluation . Several new totally artificial systems have also been described, along with the emergence of isolated hepatocyte transplantation, with reports of successful 'bridging' to liver transplantation. , , , , . Controlled trials on a multicentre basis in well-defined patient groups and with standardized outcome measures will be required to properly evaluate the clinical value of each of these approaches to providing liver support in acute liver failure and cirrhosis . A better understanding of mechanisms underlying multiorgan failure and of factors inhibiting liver regeneration, thereby allowing a more targeted approach, will be essential to the further development of effective liver support strategies in these settings.

Trends Biotechnol, 1999 Oct, 17(10), 389 - 95
Colloidal gas aphrons: potential applications in biotechnology
Jauregi P, Varley J.
Colloidal gas aphrons are microbubbles encapsulated by surfactant multilayers . They provide a large interfacial area to adsorb charged and/or hydrophobic molecules; the extent and mechanism of the adsorption depends on the surfactant multilayer . The physical properties of colloidal gas aphrons have recently been characterized for a range of surfactants in order to find the best systems for particular applications . A range of exciting biotechnology applications has been identified, including the recovery of cells, proteins and other biological molecules, and the enhancement of gas transfer in bioreactors and bioremediation.

Transgenic Res, 1999 Jun, 8(3), 237 - 47
Expression of human blood clotting factor VIII in the mammary gland of transgenic sheep; Niemann H et al.; By targeting the expression of sequences encoding non-milk proteins to the mammary gland of transgenic farm animals, the organ could serve as a 'bioreactor' for producing pharmacologically active proteins on a large scale . Here we report the generation of transgenic sheep bearing a fusion gene construct with the human blood clotting factor VIII (hFVIII) cDNA under the transcriptional control of a 2.2 kb fragment of the mammary gland specific promoter of the ovine beta-Lactoglobulin (beta-Lac) gene . Six founder animals were generated bearing a hFVIII cDNA construct with the introns of the murine metallothionein (MtI) gene (beta-Lac/hFVIII-MtI) . Founders transmitted the transgene in a Mendelian fashion and two transgenic lines were generated . Ten out of 12 transgenic F1-females expressed rhFVIII mRNA in exfoliated mammary epithelial cells isolated from the milk . But only in transgenic F1 ewes 4010 and 603 hFVIII clotting activity estimated at 4-6 ng/ml was detected in defatted milk . Furthermore, the presence of rhFVIII-protein in ovine milk was demonstrated by a specific band at approximately 190 kD following immunoprecipitation and immunoblotting . Transgenic founder 395 expressed rhFVIII mRNA in biopsied mammary gland tissue, in exfoliated mammary cells as well as ectopically in brain, heart, spleen, kidney and salivary gland, suggesting that the employed beta-Lac promoter fragment lacks essential sequences for directing expression exclusively to the mammary gland . A rhFVIII standard preparation (rhFVIIIstd) was rapidly sequestered in a saturable fashion in ovine milk, thus rendering it largely inaccessible to immunoprecipitation although its biological activity was retained . Recovery of hFVIIIstd was dependent on milk donor, storage temperature and dilution of milk sample.

Ann Biomed Eng, 1999 Jul-Aug, 27(4), 572 - 9
Estimation of the shear stress on the surface of an aortic valve leaflet; Weston MW et al.; The limited durability of xenograft heart valves and the limited supply of allografts have sparked interest in tissue engineered replacement valves . A bioreactor for tissue engineered valves must operate at conditions that optimize the biosynthetic abilities of seeded cells while promoting their adherence to the leaflet matrix . An important parameter is shear stress, which is known to influence cellular behavior and may thus be crucial in bioreactor optimization . Therefore, an accurate estimate of the shear stress on the leaflet surface would not only improve our understanding of the mechanical environment of aortic valve leaflets, but it would also aid in bioreactor design . To estimate the shear stress on the leaflet surface, two-component laser-Doppler velocimetry measurements have been conducted inside a transparent polyurethane valve with a trileaflet structure similar to the native aortic valve . Steady flow rates of 7.5, 15.0, and 22.5 L/min were examined to cover the complete range possible during the cardiac cycle . The laminar shear stresses were calculated by linear regression of four axial velocity measurements near the surface of the leaflet . The maximum shear stress recorded was 79 dyne/cm2, in agreement with boundary layer theory and previous experimental and computational studies . This study has provided a range of shear stresses to be explored in bioreactor design and has defined a maximum shear stress at which cells must remain adherent upon a tissue engineered construct.

Biotechnol Bioeng, 1999 Oct 20, 65(2), 219 - 26
Novel strategy for efficient screening and construction of host/vector systems to overproduce penicillin acylase in Escherichia coli; Chou CP et al.; A novel and simple method of using penicillin for screening of mutant strains with a high penicillin acylase (PAC) activity was developed . Random mutagenesis was conducted using a PAC-producing strain resistant to 6-aminopenicillanic acid (6-APA) as the parent strain and mutants were screened with penicillin at a high concentration . Results suggest that mutants with a high minimum inhibitory concentration for penicillin (MIC(penG)) usually overproduce PAC . Both volumetric and specific PAC activities of a mutant, MD7, were significantly higher than those of the parent strain, HBPAC101 harboring pCLL2902 . The mutation(s) resulting in the enhanced expression was mapped on the host chromosome rather than the plasmid . In addition, the mutant strain of MDDeltaP7, derived by elimination of the harbored plasmid in MD7, was demonstrated to be efficient in production of PAC by using the expression plasmids for which expression of the pac gene is limited by translation . An extremely high specific PAC activity of more than 350 U/L/OD(600) was reached upon cultivation of MDDeltaP7 harboring pTrcKnPAC2902 in a bioreactor . As such, the strategy is effective in terms of constructing PAC overproducers and improving the process yield for production of PAC .

J Vet Med Sci, 1999 Jul, 61(7), 861 - 3
Bioremediation of trichloroethylene and cis-1,2-dichloroethylene-contaminated groundwater by methane-utilizing bacteria; Arai K et al.; Experimental studies on the bioremediation of groundwater contaminated with low concentration trichloroethylene (TCE) and cis1,2-dichloroethylene (DCE) were performed with two sets of bioreactors . Reactors No . 1 and No . 2 were operated without and with methane supplement, respectively . No inoculum was used . The concentrations of TCE and DCE in the effluent and the off gas from reactor No . 2 were much lower than those from reactor No . 1 . When air and an H2O2 solution were supplied to reactor No . 2, concentrations of TCE and DCE in the effluent and the off gas were lower than the lowest detectable limit . The population of methane-utilizing bacteria in reactor No . 2 was 1,000 times higher than that in groundwater or in the effluent from reactor No . 1 . These methane-utilizing bacteria were apparently attributable to the treatment of TCE.

J Ind Microbiol Biotechnol, 1999 Jul, 23(1), 691 - 6
Kinetic models for astaxanthin production by high cell density mixotrophic culture of the microalga Haematococcus pluvialis; Zhang XW et al.; High cell density cultivation of Haematococcus pluvialis for astaxanthin production was carried out in batch and fed-batch modes in 3.7-L bioreactors with stepwise increased light intensity control mode . A high cell density of 2.65 g x L(-1) (batch culture) or 2.74 g x L(-1) (fed-batch culture) was obtained, and total astaxanthin production in the fed-batch culture (64.36 mg x L(-1)) was about 20.5% higher than in the batch culture (53.43 mg x L(-1)) . An unstructured kinetic model to describe the microalga culture system including cell growth, astaxanthin formation, as well as sodium acetate consumption was proposed . Good agreement was found between the model predictions and experimental data . The models demonstrated that the optimal light intensity for mixotrophic growth of H . pluvialis in batch or fed-batch cultures in a 3.7-L bioreactor was 90-360 micromol x m(-2) x s(-1), and that the stepwise increased light intensity mode could be replaced by a constant light intensity mode.

Curr Opin Biotechnol, 1999 Aug, 10(4), 370 - 5
Applications of oxidoreductases; May SW; Oxidoreductases comprise the large class of enzymes that catalyze biological oxidation/reduction reactions . Because many chemical and biochemical transformations involve oxidation/reduction processes, developing practical biocatalytic applications of oxidoreductases has long been an important goal in biotechnology . During the past year, significant progress has been made in the development of oxidoreductase-based diagnostic tests and improved biosensors, in the design of innovative systems for regeneration of essential coenzymes, in the construction bioreactors for biodegradation of pollutants and for biomass processing, and in the development of oxidoreductase-based approaches for synthesis of polymers and oxyfunctionalized organic substrates.

Exp Toxicol Pathol, 1999 Jul, 51(4-5), 432 - 5
Use of rat hepatocytes immobilized in agarose gel threads for biosynthesis of metabolites of potential cytostatics; Trejtnar F et al.; The aim of this work was to evaluate possibility of use of the rat isolated hepatocytes immobilized in agarose gel and continuously perfused for production of needed metabolites of two potential cytostatics, benfluron (5-(2-dimethylamino-ethoxy)-7-oxo-7H-benzo{c}fluorene) and oracin (6-{2-(hydroxyethyl) amino-ethyl}-5,11-dioxo-5,6-dihydro-11H-indeno {1,2-c}isoquinoline) . The rat isolated hepatocytes obtained by two-step collagenase perfusion method were immobilized in agarose threads and perfused in a small bioreactor under a recirculation regimes . Biosynthesis of 9-hydroxybenfluron and 3-hydroxyoracin in immobilized rat hepatocytes was studied . Yields of the metabolites of interest in hepatocytes in immobilized and perfused rat hepatocytes was compared to production of metabolites in hepatocyte suspension and in rats in vivo . 9-hydroxybenfluron was presented during perfusion of immobilized rat hepatocytes in a relatively high amounts but total recovery all forms of benfluron was very low due to especially high binding to components of the perfusion system . More effective method remains the production of 9-hydroxybenfluron in rats in vivo . A considerable biosynthesis of 3-hydroxyoracin by immobilized rat hepatocytes in the bioreactor was found . Concentration of the metabolite in the perfusate rose continuously during 6 hours of perfusion . 3-hydroxyoracin production was increased several times with use of immobilized hepatocytes from rats treated for three days with methylcholanthrene . The yield of 3-hydroxyoracin in rats in vivo was comparably high but an advantage of in vitro synthesis is a much shorter interval to obtain the same amount of the metabolite of interest . In spite of some limitations in compounds exerting high trapping in the perfusion system, the method of the immobilized and perfused hepatocytes can be very useful and effective for production of some drug metabolites in biochemistry and pharmacology.

Am J Physiol, 1999 Aug, 277(2 Pt 2), H433 - 44
Cardiac muscle tissue engineering: toward an in vitro model for electrophysiological studies; Bursac N et al.; The objective of this study was to establish a three-dimensional (3-D) in vitro model system of cardiac muscle for electrophysiological studies . Primary neonatal rat ventricular cells containing lower or higher fractions of cardiac myocytes were cultured on polymeric scaffolds in bioreactors to form regular or enriched cardiac muscle constructs, respectively . After 1 wk, all constructs contained a peripheral tissue-like region (50-70 micrometer thick) in which differentiated cardiac myocytes were organized in multiple layers in a 3-D configuration . Indexes of cell size (protein/DNA) and metabolic activity (tetrazolium conversion/DNA) were similar for constructs and neonatal rat ventricles . Electrophysiological studies conducted using a linear array of extracellular electrodes showed that the peripheral region of constructs exhibited relatively homogeneous electrical properties and sustained macroscopically continuous impulse propagation on a centimeter-size scale . Electrophysiological properties of enriched constructs were superior to those of regular constructs but inferior to those of native ventricles . These results demonstrate that 3-D cardiac muscle constructs can be engineered with cardiac-specific structural and electrophysiological properties and used for in vitro impulse propagation studies.

Biotechnol Prog, 1999 Jul-Aug, 15(4), 630 - 9
Development and validation of a methodology for intracellular pH measurements of hybridoma cells under bioreactor culture conditions; Cherlet M et al.; The intracellular pH (pH(i)) is an important factor in the regulation of different cellular processes . It might therefore be used as a marker of the physiological state of cells cultivated in a bioreactor environment . We developed and validated therefore a methodology that permits a reproducible and reliable pH(i) measurement under such bioreactor culture conditions, contrary to earlier reported measurements, carried out on cells resuspended in buffers under nongrowth conditions . The hybridoma cells were sampled from the culture, stained with the pH-sensitive dye BCECF-AM (BCECF = 2',7'-bis-carboxyethyl-5,6-carboxyfluorescein), and analyzed by flow cytometry . Such a measurement is perfectible to changes of the cells between the moment of sampling and of final analysis on the flow cytometer . All intermittent steps were for this reason studied in detail, either to determine the optimal conditions to be used or to characterize their influence on the final pH(i) value measured . Additional experiments were carried out, showing the representativeness of the measured pH(i) value for the pH(i) the cells possess really in the culture at the moment of sampling.

Biotechnol Bioeng, 1999 Oct 5, 65(1), 44 - 53
The effect of temperature on the continuous ferrous-iron oxidation kinetics of a predominantly Leptospirillum ferrooxidans culture; Breed AW et al.; The ferrous-iron oxidation kinetics of a bacterial culture consisting predominantly of Leptospirillum ferrooxidans were studied in continuous-flow bioreactors . The bacterial culture was fed with a salts solution containing 12 g/L ferrous-iron, at dilution rates ranging from 0.01 to 0.06 l/h, and temperatures ranging from 30 to 40 degrees C, at a pH of 1.75 . The growth rate, and the oxygen and ferrous-iron utilization rates of the bacteria, were monitored by means of off-gas analysis and redox-potential measurement . The degree-of-reduction balance was used to compare the theoretical and experimental values of r(CO(2)), -r(O(2)) and -r(Fe(+2)), and the correlation found to be good . The maximum bacterial yield on ferrous-iron and the maintenance coefficient on ferrous-iron, were determined using the Pirt equation . An increase in the temperature from 30 to 40 degrees C did not appear to have an effect on either the maximum yield or maintenance coefficient on ferrous-iron . The average maximum bacterial yield and maintenance coefficient on ferrous-iron were found to be 0.0059 mmol C/mmol Fe(2+) and 0.7970 mmol Fe(2+)/mmol C)/h, respectively . The maximum specific growth rate was found to be 0.077 l/h . The maximum specific ferrous-iron utilization rate increased from 8.65 to 13.58 mmol Fe(2+)/mmol C/h across the range from 30 to 40 degrees C, and could be described using the Arrhenius equation . The kinetic constant in bacterial ferrous-iron oxidation increased linearly with increasing temperature . The ferrous-iron kinetics could be accurately described in terms of the ferric/ferrous-iron ratio by means of a Michaelis-Menten-based model modified to account for the effect of temperature . A threshold ferrous-iron level, below which no further ferrous-iron utilization occurred, was found at a ferric/ferrous-iron ratio of about 2500 . At an overall iron concentration of 12 g/L, this value corresponds to a threshold ferrous-iron concentration of 78.5 x10(-3) mM .

Biotechnol Bioeng, 1999 Oct 5, 65(1), 17 - 23
Immobilization of recombinant heparinase I fused to cellulose-binding domain; Shpigel E et al.; Immobilization of biologically active proteins is of great importance to research and industry . Cellulose is an attractive matrix and cellulose-binding domain (CBD) an excellent affinity tag protein for the purification and immobilization of many of these proteins . We constructed two vectors to enable the cloning and expression of proteins fused to the N- or C-terminus of CBD . Their usefulness was demonstrated by fusing the heparin-degrading protein heparinase I to CBD (CBD-HepI and HepI-CBD) . The fusion proteins were over-expressed in Escherichia coli under the control of a T7 promoter and found to accumulate in inclusion bodies . The inclusion bodies were recovered by centrifugation, the proteins were refolded and recovered on a cellulose column . The bifunctional fusion protein retained its abilities to bind to cellulose and degrade heparin . C-terminal fusion of heparinase I to CBD was somewhat superior to N-terminal fusion: Although specific activities in solution were comparable, the latter exhibited impaired binding capacity to cellulose . CBD-HepI-cellulose bioreactor was operated continuously and degraded heparin for over 40 h without any significant loss of activity . By varying the flow rate, the mean molecular weight of the heparin oligosaccharide produced could be controlled . The molecular weight distribution profiles, obtained from heparin depolymerization by free heparinase I, free CBD-HepI, and cellulose-immobilized CBD-HepI, were compared . The profiles obtained by free heparinase I and CBD-HepI were indistinguishable, however, immobilized CBD-HepI produced much lower molecular weight fragments at the same percentage of depolymerization . Thus, CBD can be used for the efficient production of bioreactors, combining purification and immobilization into essentially a single step .

Zhonghua Nei Ke Za Zhi, 1997 Oct, 36(10), 665 - 8
{Establishment and significance of an extracorporeal bioartificial liver support system}; Wang Y et al.; This experiment was carried out to establish an optimal extracorporeal bioartificial liver support system (EBLSS) and furnish a new therapy for hepatic failure . For this purpose, human hepatocytes were isolated by two-stage perfusion method and cultured by spheroidal aggregates method . Besides, hollow fiber tube was used as bioreactor and the hemodialysis instrument was refited to serve as supplementary circulation unit, which was composed of EBLSS and was tried in normal dogs . A total of 2 x 10(10) human hepatocytes with a viability of 94% was obtained . These hepatocytes became multicellular spheroidal aggregates under non-adherent conditions . After 5 hours circulating on EBLSS, the hepatocytes remained active . At the same time, the monitoring system revealed a normal vital sign in dogs and the laboratory tests found their serum total protein and albumin were increased . These results show that the key technology of bioartificial liver has been dereloped basically and this EBLSS is effective and safe, providing a foundation for the support and treatment of hepatic failure with bioartificial liver.

Ther Apher, 1999 Aug, 3(3), 264 - 7
A new concept of bioartificial liver based on a fluidized bed bioreactor; Dore E et al.; Many bioartificial livers have been developed, but most of them suffer from difficulty when being scaled up and from poor efficiency of mass transfer between the plasma and the immobilized hepatocytes . We present a new concept of bioartificial liver based on the fluidized bed motion of hepatocytes entrapped in alginate beads . The bioreactor is designed to offer stable behavior . The maximum fluid perfusion velocity is determined to avoid any bead release from the bioreactor . The fluidized bed height depends on the amount of beads and the velocity employed . Under the optimized operating conditions, the mass transfer between perfusion fluid and beads is very efficient; only 10 min are necessary to reach concentration equilibrium . Hence, this fluidized bed bioartificial liver appears to be a promising tool for a liver support system in the treatment of acute liver failure.

Artif Cells Blood Substit Immobil Biotechnol, 1999 Jul, 27(4), 313 - 42
Dual enzyme multi-layer bioreactors: analytical modeling and experimental studies; Segal V et al.; Enzymic reactors are developed for a variety of biomedical-biotechnological applications, including blood detoxification . For the latter, an appropriate approach is to use enzymes of the Mercapturic Acid Pathway . The first two enzymes of this pathway are Glutathione-S-Transferase (GST) and gamma-Glutamyl Transpeptidase (gamma GT) . Earlier, the performance of an immobilized GST reactor was investigated experimentally and theoretically . Here, the analytical model was extended to describe a dual-enzyme continuous packed-bed reactor (DCP), in which the two enzymes (E1 and E2) are arranged in alternating layers . The performance of DCP reactors was first studied by numerical simulations, considering the effects of reactor configuration (i.e . number of enzyme layers), kinetic characteristics (K(m), Vmax, Kiq) and operational parameters (flow rate, substrates concentration) . Results were obtained in terms of substrate and products concentration profiles along the reactor . The theoretical calculation were supplemented by experimental studies . In the latter GST (i.e . E1) and gamma GT (i.e . E2), were used when immobilized on porous beads, and the reactor was set up and operated in various configurations . It was found that the factors which mostly affect the performance of DCP systems are reactor configuration and extent of inhibition of E1 by its reaction product.

Int J Oncol, 1999 Sep, 15(3), 511 - 8
Production of ex vivo expanded hematopoietic cells and progenitors in a closed bioreactor, starting with a small volume marrow collection: A feasibility study in patients with poor-risk breast cancer and receiving high-doses of cyclophosphamide; Chabannon C et al.; We report a clinical pilot study conducted in 6 women with poor-prognosis breast cancer . The goal was to evaluate the feasibility and safety of producing hematopoietic progenitors and cells from a small marrow sample, for clinical use after high-dose cyclophosphamide . A small volume marrow collection was obtained, using local anesthesia and conscious sedation, before the first of two chemotherapy cycles . Cells were cryopreserved, and later thawed to inoculate two Aastrom Biosciences Inc Replicell bioreactors, on time to reinfuse ex vivo expanded cells after the second chemotherapy cycle . Patients recovered neutrophils and platelets at similar times after the first and second chemotherapy cycles, and showed comparable clinical events . This pilot study prepares future randomized trials, designed to evaluate clinical benefits associated with the use of ex vivo expanded cells in the setting of multicycle high-dose chemotherapy.

Appl Environ Microbiol, 1999 Aug, 65(8), 3750 - 3
Production of a new D-amino acid oxidase from the fungus Fusarium oxysporum; Gabler M et al.; The fungus Fusarium oxysporum produced a D-amino acid oxidase (EC 1 . 4.3.3) in a medium containing glucose as the carbon and energy source and ammonium sulfate as the nitrogen source . The specific D-amino acid oxidase activity was increased up to 12.5-fold with various D-amino acids or their corresponding derivatives as inducers . The best inducers were D-alanine (2.7 microkat/g of dry biomass) and D-3-aminobutyric acid (2.6 microkat/g of dry biomass) . The addition of zinc ions was necessary to permit the induction of peroxisomal D-amino acid oxidase . Bioreactor cultivations were performed on a 50-liter scale, yielding a volumetric D-amino acid oxidase activity of 17 microkat liter(-1) with D-alanine as an inducer . Under oxygen limitation, the volumetric activity was increased threefold to 54 microkat liter(-1) (3,240 U liter(-1)).

Appl Environ Microbiol, 1999 Aug, 65(8), 3265 - 71
Engineering of stable recombinant bacteria for production of chiral medium-chain-length poly-3-hydroxyalkanoates; Prieto MA et al.; In order to scale up medium-chain-length polyhydroxyalkanoate (mcl-PHA) production in recombinant microorganisms, we generated and investigated different recombinant bacteria containing a stable regulated expression system for phaC1, which encodes one of the mcl-PHA polymerases of Pseudomonas oleovorans . We used the mini-Tn5 system as a tool to construct Escherichia coli 193MC1 and P . oleovorans POMC1, which had stable antibiotic resistance and PHA production phenotypes when they were cultured in a bioreactor in the absence of antibiotic selection . The molecular weight and the polydispersity index of the polymer varied, depending on the inducer level . E . coli 193MC1 produced considerably shorter polyesters than P . oleovorans produced; the weight average molecular weight ranged from 67,000 to 70,000, and the polydispersity index was 2.7 . Lower amounts of inducer added to the media shifted the molecular weight to a higher value and resulted in a broader molecular mass distribution . In addition, we found that E . coli 193MC1 incorporated exclusively the R configuration of the 3-hydroxyoctanoate monomer into the polymer, which corroborated the enantioselectivity of the PhaC1 polymerase enzyme.

Moisture content is the single most important factor that promotes the accelerated decomposition. The bioreactor technology relies on maintaining optimal moisture content near field capacity (approximately 35 to 65%) and adds liquids when it is necessary to maintain that percentage. The moisture content, combined with the biological action of naturally occurring microbes decomposes the waste. The microbes can be either aerobic or anaerobic. A side effect of the bioreactor is that it produces landfill gas (LFG) such as methane in an anaerobic unit at an earlier stage in the landfill’s life and at an overall much higher rate of generation than traditional landfills.

In batch cultivation, an innoculum of known density is seeded into a specified volume of pre-conditioned medium in the bioreactor. Ideally nothing is added or removed from the bioreactor during the course of cultivation. However in practice additions of air and acids or bases for pH control are made. Batch cultivation of suspension cells can be carried out in two types of bioreactors: Stirred tank and air lift reactors.

In airlift bioreactors for hybridoma cultivation, gas is introduced at the bottom of the vessel within the draught tube. A reduction in density of the aerated contents in the draught tube results in a circulation of the culture through the draught tube and down in the outer zone of the vessel. Advantages are that there are no moving parts or mechanical seals, there is adequate oxygen transfer, low hydrodynamic shear forces and low power input per unit volume. Operation: Batch operation with temperature controlled at 37 OC via a cooling jacket. pH is controlled by automatic addition of CO2 into the sparged gas or by NaOH. Dissolved oxygen tension is controlled by varying the concentration of oxygen in air. Foaming may be controlled by the addition of antifoam agent in conjunction with pluronic F-68. Scale Up: 10-2000L airlift bioreactors are used for cultivation of hybridoma cultures. Improved oxygen transfer rates have been shown at the larger scales. The increase in hydrostatic pressure resulting from increased bioreactor height does not have a deleterious effect on the cells. Media: Media supplement with 2-10 % animal sera as well as defined serum free media have been successfully used. Hybridoma cell lines of mouse, rat and human origin have been cultivated batch wise for 10 –17 days depending on the particular cell line. Hybridoma growth kinetics is not affected by dissolved oxygen tension in the range of 10 – 100 % saturation. Foaming is a problem at small scales and leads to the loss of cells and product.

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