Bioreactor

Can J Physiol Pharmacol, 1994 Apr, 72(4), 407 - 14
Digestion and absorption of food: usefulness and limitations of in vitro models; Savoie L; The digestion and absorption of food is a spatiotemporal and dynamic process involving complex enzymatic and transport reactions, and it is illusive to try to reproduce in a single model all these biochemical and physiological events . A more practical and realistic approach is to separately evaluate the specific contributions of oral and gastric digestion, intestinal digestion by pancreatic enzymes, brush-border hydrolysis, and eventually intestinal absorption and enterocyte metabolism . The models proposed must be versatile enough to be able to modify their conditions of operation according to physiological adaptation to food . Enzymatic preparations must be kept close to physiological conditions in regard to their nature and their mode of operation . A digestion cell and a peptidase bioreactor were developed for this purpose . The challenge is to find a way to integrate all these data . This can be partially achieved by selecting techniques that allow the collection and isolation of reaction products from one step for use as substrates for the next event . Various models are presented to illustrate this concept as applied to food protein.

Glycoconj J, 1994 Apr, 11(2), 153 - 62
The ganglioside GD1 alpha' IV3Neu5Ac, III6Neu5Ac-GgOse4Cer, is a major disialoganglioside in the highly metastatic murine lymphoreticular tumour cell line MDAY-D2; Muthing J et al.; The aim of the present study was to investigate the ganglioside expression of the highly metastatic murine lymphoreticular tumour cell line MDAY-D2 . Cells were propagated under controlled pH conditions and oxygen supply in bioreactors of 1 and 7.5 l volumes by repeated batch fermentation . Gangliosides were isolated from 2.7 x 10(11) cells, purified by silica gel chromatography and separated into mono- and disialoganglioside fractions by preparative DEAE anion exchange high performance liquid chromatography . Individual gangliosides were obtained by preparative thin layer chromatography . Their structural features were established by immunostaining, fast atom bombardment and gas chromatography mass spectrometry . In addition to gangliosides of the GM1a-pathway (GM2, GM1a and GD1a) and GM1b (IV3Neu5Ac-GgOse4Cer) and GalNAc-GM1b of the Gm1b-pathway, the disialoganglioside GD1 alpha (IV3Neu5Ac, III6Neu5Ac-GgOse4Cer) was found in equal amounts compared to GD1a (IV3Neu5Ac, II3Neu5Ac-GgOse4Cer) . All gangliosides were substituted with C24:0, 24:1 and C16:0 fatty acids, sphingosine and N-acetylneuraminic acid as the sole sialic acid.

J Biotechnol, 1994 Mar 31, 33(2), 107 - 22
Unified modeling framework of cell death due to bubbles in agitated and sparged bioreactors; Wang NS et al.; A modeling framework is proposed to assess the detrimental effects of air sparging and other bubble phenomena (vortex entrainment, coalescence, bursting) on freely suspended cells in an aerated, agitated bioreactor . It is assumed that cells may be rendered nonviable by bubble breakup/coalescence within the medium, by bubble formation at the sparger, or by bubble bursting at the free surface . Some plausible mechanisms are argued from the energetic view point . The dominant parameters in each case are the cell-bubble encounter rate and the bubble breakup/bursting rate . These inactivation processes lead to a Michaelis-Menten expression for the specific cell death rate, which is shown to be linearly proportional to the specific bubble interfacial area (total bubble surface area per unit volume of media) . By using published viable cell concentration data for retarded growth of mammalian cells due to sparging, the interfacial area correlation is demonstrated . The method is generalized to aerated bioreactor conditions . The article offers a unique, consistent perspective on how cell death can be viewed.

J Biotechnol, 1994 Mar 15, 33(1), 21 - 31
Intracellular calcium response of Sf-9 insect cells exposed to intense fluid forces; Aloi LE et al.; Suspension cell cultures are exposed to periodic high intensity, short duration fluid forces by circulating them through a flow loop containing a capillary, which simulates what a cell experiences in a stirred bioreactor . Sf-9 insect cells exhibit an increase in intracellular calcium concentration, {Ca2+}i when exposed to these cyclic fluid forces . Flow through the capillary spans both the laminar and turbulent regime and the calcium response is not dependent on the transition to turbulence . The calcium response is a nearly linear function of the rate of energy dissipation per mass of fluid in the capillary . The source of the increased calcium ions in the cell cytosol is within the cell itself, indicating that the calcium response is a cellular response to fluid forces and not a matter of increased plasma membrane permeability to Ca2+ ions . Flow cytometry on hydrodynamically stimulated and unstimulated cells reveals that the increase in the intracellular calcium concentration averaged over the cell population is due to an increase in intracellular calcium concentration in only a small sub-population of the entire suspension.

Artif Organs, 1994 Mar, 18(3), 226 - 30
Hepatocyte culture between woven capillary networks: a microscopy study; Gerlach J et al.; A multi-compartment capillary membrane culture model with independently perfused three-dimensionally woven capillaries was developed for immobilization of hepatocytes in bioreactors . This enables spatial restructuring of cells and enhanced mass transfer performance with more efficient oxygenation and metabolite exchange . Seeding density defines cell behaviour in this model . With low densities cells attach to the membranes and flatten . Increasing density leads to spontaneous formation of aggregates which are immobilized between the capillaries.

Biodegradation, 1994 Mar, 5(1), 1 - 11
Optimization and maintenance of soluble methane monooxygenase activity in Methylosinus trichosporium OB3b; Bowman JP et al.; Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater . A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity in Methylosinus trichosporium OB3b . Methylosinus trichosporium OB3b attained peak sMMO activity (275-300 nmol of naphthol formed h-1 mg of protein-1 at 25 degrees C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium . With the onset of methane limitation however, sMMO activity rapidly declined . It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (mu max 0.08 h-1) and growth yields (0.4-0.5 g cells/g CH4) and near maximal activities of sMMO . In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45-50% of the initial peak level and this was maintained over several weeks . The addition of d-biotin, pyridoxine, and vitamin B12 (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25-75% . The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.

J Chem Technol Biotechnol, 1994 Mar, 59(3), 297 - 302
Residence time distribution in a packed bed bioreactor containing porous glass particles: influence of the presence of immobilized cells; De Backer L et al.; An experimental investigation of the liquid phase residence time distribution (RTD) in a packed bed bioreactor containing porous glass particles is presented . For Re < 1, intraparticle forced convection is negligible and only diffusion, characterized by an effective diffusion coefficient, must be considered to describe the mass transfer process between the extraparticle and the intraparticle fluid phase . For Re > 1, the mass transfer rate becomes dependent on the liquid flow rate, indicating the existence of intraparticle convection . A model including axially dispersed flow for the external fluid phase and an 'apparent' effective diffusivity that combines diffusion and convection, predicts experimental RTD data satisfactorily . Yeast cells immobilized inside the porous glass beads did not affect the mass transfer rate at low biomass loading . At high biomass loading (0.02 g yeast cells g-1 carrier), the mass transfer rate between the extraparticle and intraparticle fluid phase was significantly decreased . Comparison of the RTD data from experiments performed in the presence and absence of cells in the external fluid phase revealed that the mass transfer rate is influenced by the cells immobilized inside the porous particles and not by the cells present in the external fluid phase.

Biotechnol Prog, 1994 Mar-Apr, 10(2), 198 - 206
Viable cell recycle with an inclined settler in the perfusion culture of suspended recombinant Chinese hamster ovary cells; Searles JA et al.; The perfusion culture of suspended mammalian cells requires a cell retention device, the best of which will retain all viable cells and reject all nonviable cells and debris . The inclined settler is a passive, simple, inexpensive, and easy-to-maintain device that has been shown in the past to selectively remove single nonviable cells of hybridoma cultures . In this work, we have demonstrated the preferential return of viable recombinant Chinese hamster ovary (CHO) cells through the use of a three-port settler maintained at lower temperatures and vibrated to reduce cell attachment and enhance cell return to the bioreactor . The residence time of CHO cells in the cooled, vibrated settler was determined by flow-cytometric discrimination of tracer recombinant CHO cells . Cells returning to the bioreactor through the underflow had an average residence time of 1.46 h in the settler . During perfusion cultures with cell densities above 10(6) cells/mL, cells seen to be stalled within the settler were easily dislodged by periodic air bubbling using a simple back-flushing procedure in which headspace gas was brought through the settler underflow port . The resuspended cells were returned to the bioreactor within an average of 32 min after bubbling . This study demonstrates that inclined sedimentation technology can be utilized to selectively recycle viable recombinant CHO cells with only a short retention time in an inclined settler.

Enzyme Microb Technol, 1994 Mar, 16(3), 253 - 7
Interaction of transport resistances with biochemical reaction in packed-bed solid-state fermentors: effect of temperature gradients; Ghildyal NP et al.; In solid-state fermentation, the interaction of transport phenomena with biochemical reactions has a considerable effect on the productivity of the bioreactor . Previous work on solid-state fermentation in tray fermentors in our laboratory indicated that heat transfer resistance results in steep temperature gradients within the solid substrate bed, which in turn adversely affect the biochemical reaction and enzyme activity . This problem of heat accumulation during the course of fermentation has been alleviated to a considerable extent using a packed-column bioreactor with forced aeration in the present work . Experimental studies were conducted in a packed-column bioreactor utilizing wheat bran as substrate and the fungus Aspergillus niger CFTRI 1105 for the production of the enzyme amyloglucosidase . The enzyme activities were estimated and temperatures were recorded at different bed heights, for different air flow rates during the course of fermentation . The results indicated that the temperature gradients caused by heat transfer resistances were reduced considerably with corresponding increases in enzyme activity.

Biotechnology (N Y), 1994 Mar, 12(3), 281 - 4
Acoustic cell filter for high density perfusion culture of hybridoma cells; Trampler F et al.; We have developed a flow-through device which uses high frequency, low energy ultrasonic resonance fields to transiently aggregate hybridoma cells and return them by sedimentation to a perfusion bioreactor . The system retained up to 99 percent of the inflowing viable cells with no measurable effect on viability . Viable cells were selectively retained at up to 3 percent higher efficiency than nonviable cells . A stirred tank bioreactor was operated for 700 hours with acoustic cell recycle . Concentrations greater than 5 x 10(7) cells/ml were attained with a 5-fold increase in antibody concentration and a 70-fold increase in volumetric productivity compared with batch culture.

J Biotechnol, 1994 Feb 28, 32(3), 231 - 8
Expression of alpha 1-proteinase inhibitor in Escherichia coli: effects of single amino acid substitutions in the active site loop on aggregate formation; Schulze AJ et al.; Overproduction of eukaryotic proteins in microorganisms often leads to the formation of insoluble protein aggregates which accumulate as intracellular inclusion bodies . alpha 1-Proteinase inhibitor (alpha 1-PI) when produced as a cytoplasmic protein in Escherichia coli (E . coli) forms inclusion bodies containing the majority of the inhibitor in an inactive form . Several variants of alpha 1-PI with single amino acid substitutions within their active site loop (amino acids 345-358) were produced in a bioreactor showing that substitution of Met351 with Glu resulted in significantly reduced aggregate formation compared to the other variants and to wild-type protein . In addition, this variant proved to be fully functional as a proteinase inhibitor . Based on these findings and on results of previous structural studies a mechanism for aggregate formation during expression of alpha 1-PI is suggested.

J Biotechnol, 1994 Feb 14, 32(2), 127 - 38
Optical triple sensor for measuring pH, oxygen and carbon dioxide; Weigl BH et al.; A triple sensor unit consisting of opto-chemical sensors for measurement of pH, oxygen and carbon dioxide in bioreactors is presented . The pH and the CO2 sensor are based on the color change of a pH-sensitive dye immobilized on a polymeric support . The resulting changes in absorption are monitored through optical fibers . The oxygen sensor is based on the quenching of the fluorescence of a metal-organic dye . All three sensors are fully LED compatible . The sensitive membranes consist of plastic films and can be stored and replaced conveniently . The sensors are sterilizable with hydrogen peroxide and ethanol . In addition, the pH sensor is steam sterilizable . Accuracy, resolution and reproducibility fulfill the requirements for use in biotechnological applications . Calibration procedures for each sensor are presented . The working principle and the performance of all three sensors are described, with particular emphasis given to their application in bioreactors.

Appl Biochem Biotechnol, 1994 Feb, 44(2), 151 - 60
Immobilized carboxypeptidase N . A potent bioreactor and specific adsorbent for peptides; Wang W et al.; Carboxypeptidase N-Sepharose was prepared by covalent immobilization of purified human plasma carboxypeptidase N . More than 98% of the carboxypeptidase N was immobilized; 42% of the applied activity can be detected on the support . The column has excellent capabilities to quantitatively remove carboxy-terminal basic amino acids from peptides, as is demonstrated using the synthetic peptide substrate hippuryl-L-arginine and the nonapeptide bradykinin, and remains stable for several months . In contrast with apocarboxypeptidase B-Sepharose, apocarboxypeptidase N-Sepharose poorly binds its substrates.

Enzyme Microb Technol, 1994 Feb, 16(2), 151 - 8
A new enzyme immobilization procedure using copper alginate gel: application to a fungal phenol oxidase; Palmieri G et al.; A new procedure was developed for enzyme immobilization by entrapment in copper alginate gel . The mechanical properties of the copper alginate gel were characterized and compared with those of the most widely used calcium alginate . The system was applied to the immobilization of a fungal phenol oxidase . Optimal conditions for enzyme immobilization were set up: the system immobilized 85% of the enzyme, and the remaining 15% was recovered in the aqueous immobilization medium . The stability and activity of the immobilized enzyme were studied . After immobilization, the enzyme was active in a wider pH range, the temperature of its optimal activity was shifted to lower values, and the possibility of storage at 4 degrees C was greatly improved . The immobilized enzyme generally increased the rate of oxidation of various substrates . The results indicate a potential use of this system for the construction of bioreactors to be used in the detoxification of polluted waste waters.

J Immunol Methods, 1994 Jan 3, 167(1-2), 109 - 19
Enhanced IgG production in eRDF media with and without serum . A comparative study; Chua F et al.; The performance of three basal media RPMI, DMEM/F12 (DF) and eRDF (enhanced RDF, RPMI:DMEM:F12 in 2:1:1) were evaluated in cultures with and without serum with respect to cell proliferation, metabolism and monoclonal antibody (Mab) productivity . Based on the ease of adaptation, growth rate, maximum cell density and Mab production, the media were ranked as follows: eRDF > DF > RPMI . This was true for serum-free (SF) and serum supplemented (SS) media in static and shaker cultures . Growth performances in static and shaker cultures were consistently 20-50% lower in all three SF media compared to the corresponding SS conditions . Antibody titres in DF/SF and RPMI/SF cultures, irrespective of the culture condition, were generally similar or slightly lower than their SS counterparts . However, eRDF/SF medium yielded a much higher Mab titre (193 mg l-1) compared to eRDF/SS medium (145 mg l-1) . This was also six times higher than the lowest titre of 30 mg l-1 in RPMI/SF medium . Hybridomas in eRDF/SF were further adapted to media without bovine serum albumin (eRDF/SF-BSA) . Maximum cell densities in these cultures improved with scale up, from 1.1 x 10(6) ml-1 in static, to 1.9 x 10(6) ml-1 in shaker flasks, to 2.5 x 10(6) ml-1 in bioreactors . However, Ig levels remained between 100-130 mg l-1 which were much lower than in eRDF/SF medium . Thus BSA appears to be necessary for Ig production . The manufacturing cost (excluding purification) of Ig using eRDF was calculated to be between 17-50% of the price of the other two media and therefore this is regarded as the best medium for Ig production.

In Vitro Cell Dev Biol Anim, 1994 Jan, 30A(1), 23 - 9
Primary cultures of rat hepatocytes in hollow fiber chambers; Jauregui HO et al.; Hepatocyte culture may represent an alternative to the use of animals to study drug detoxification by the liver . An ideal in vitro system should closely mimic the in vivo environment by providing continuous media perfusion and oxygenation, and should facilitate sampling of cells and culture media . To meet these criteria, a hollow fiber bioreactor seeded with isolated rat hepatocytes was developed and tested by measuring the formation of three products of the oxidative metabolism of diazepam and the glucuronidation of phenolsulfonphthalein (PSP) . To compare the performance of conventional monolayer culture to that of the bioreactor system, diazepam metabolism was studied for 45 days in both systems . The oxygen dependency of diazepam metabolism was evaluated by perfusing the bioreactor in an oxygen-rich atmosphere (30%) . Total diazepam metabolism was twofold higher in the O2-rich perfused hollow fiber cultures than in the cultures perfused under normal conditions, reflecting an increase in temazepam and oxazepam production . Diazepam detoxification activity was significantly enhanced by oxygen (P < or = 0.001) over the life of the perfused cultures . PSP metabolism was similar in all three culture systems . By Day 10, diazepam metabolism in the oxygenated bioreactor system was 44% of the in vivo activity of rat hepatocytes . This activity dropped to 30% by Day 25 of culture . These results justify the use of perfused culture systems for in vitro detoxification studies as an alternative to animal use and emphasize the capacity of a culture device perfused under O2-enriched conditions to maintain long-term P450 activity of rat hepatocytes.

Hum Gene Ther, 1994 Jan, 5(1), 19 - 28
Improved methods of retroviral vector transduction and production for gene therapy; Kotani H et al.; To facilitate clinical applications of retroviral-mediated human gene transfer, retroviral vectors must be of high titer and free of detectable replication-competent retroviruses . The purpose of this study was to optimize methods of retroviral vector production and transduction . Studies were conducted using 22 retroviral vector producer cell lines . Inactivation of retroviral vectors was greater at 37 degrees C than at 32 degrees C . A 5- to 15-fold increase of vectors was produced at 32 degrees C compared to 37 degrees C; the vector increase at 34 degrees C was intermediate . For example, PA317/G1Na.40 grew to a titer of 1.8 x 10(7) cfu/ml at 32 degrees C, compared to 5.0 x 10(5) cfu/ml at 37 degrees C . The production of retroviral vectors was scalable achieving similar results in flasks, roller bottles, or a CellCube Bioreactor . Retroviral vectors were concentrated 15-24 times with vector recovery ranging from 91 to 96% in a Pellicon tangential flow filtration system . Retroviral supernatants were successfully lyophilized . The combination of glucose or sorbitol with gelatin resulted in recovery rates of 64-83% . In studies on transduction by retroviral vectors, centrifugation of vector supernatants onto target cells significantly increased transduction efficiency as measured by vector titration for G418 resistance, fluorescence-activated cell sorting (FACS), and polymerase chain reaction (PCR) analyses . The combination of the above methods has significantly increased the growth and transduction by this vector system.

Res Microbiol, 1994 Jan, 145(1), 49 - 52
Disposal of slop oil and sludges by biodegradation; Jack TR et al.; These pilot tests indicate that immobilized microbial populations can degrade a wide range of crude oils absorbed into a properly prepared peat matrix with surprising speed and flexibility . Depending on the hydrocarbon composition, ultimate disposal by landfarming or landfilling of the residues is possible . In the former case, performance of the landfarm will be enhanced and environmental concerns related to its operation reduced . In terms of ease of operation, capital and operating costs and reduced environmental concerns, the novel bioreactor presented here meets the requirements and cost constraints associated with on-site slop oil and sludge disposal for the sorts of volumes normally encountered . A patent has been applied for (Francis and Jack, 1991) . The technological issues in this development process largely arose from requirements and constraints associated with the target application . Scientific issues surrounding the enhanced biodegradation, seen especially for absorbed heavy oil, remain unresolved . These may or may not be picked up in further development and optimization as need and cost allow.

Crit Rev Biotechnol, 1994, 14(2), 75 - 107
Immobilization of cells for application in the food industry; Groboillot A et al.; Immobilization of cells offers advantages to the food process industries, including enhanced fermentation productivity and cell stability and reduced downstream processing costs due to facilitated cell recovery and recycle . This article summarizes the varied immobilization methodologies, including adsorption, entrapment, covalent binding, and microencapsulation . Examples of interest to the food industry are provided, together with a review of the physiological effects of immobilization . Topics in process engineering include immobilized cell bioreactor configurations and the scale-up potential of the various immobilization techniques.

Zentralbl Chir, 1994, 119(5), 334 - 40
{Cell culture model for hepatocyte culture in bioreactors for metabolic utilization in hybrid liver support system}; Gerlach J et al.; Hybrid systems with hepatocyte cultures in bioreactors are in development for therapeutical liver assistance . Here, a culture model with a special bioreactor construction was developed: Capillary membrane systems create a three dimensional artificial framework for hepatocyte adhesion, aggregation and reorganization of tissue structures . Many of small parallel capillary membrane units perfuse a few hepatocytes . Different capillary materials enable different functions . Cell perfusion between independent plasma inflow and outflow capillaries, independent oxygen supply and carbon dioxide removal as well as a co- culture with sinusoidal endothelial cells are possible . An in vitro study with bioreactors (n = 9), containing 2.5 x 10(9) pig hepatocytes was performed, measuring external metabolism of the cells: cytochrome P450-activity (midazolam metabolism, lidocaine/MEGX-test), synthesis (albumin), liver function test (galactose elimination) and cell alteration (LDH, GOT, GLDH, GPT, gamma GT) were investigated . The results demonstrate that external function of primary hepatocytes can be maintained over a period of at least three weeks.

Biochem Mol Biol Int, 1994 Jan, 32(1), 87 - 94
Interaction of cationic phospholipid vesicles with carbonic anhydrase; Annesini MC et al.; The possibility of entrapping the enzyme carbonic anhydrase into liposomes, in order to obtain small, membrane-confined bioreactors for biotechnological or biomedical applications, was studied . Neutral liposomes (dipalmitoylphosphatidylcholine/cholesterol) or cationic liposomes (dipalmitoylphosphatidylcholine/cholesterol/stearylamine) with different dipalmitoylphosphatidylcholine/stearylamine ratios have been used to trap carbonic anhydrase . Kinetic experiments showed that carbonic anhydrase was being trapped into cationic liposomes, but not into neutral ones . A significant amount of carbonic anhydrase was sitting onto the external surface of liposomes when the ratio dipalmitoylphosphatidylcholine/cholesterol/stearylamine was 6:3:1, but not when it was 5:3:2 . Morphological analysis by electron microscopy showed that the presence of carbonic anhydrase induced a significant swelling in the 6:3:1 cationic vesicles, related to the activity of the enzyme.

Bioelectromagnetics, 1994, 15(4), 303 - 13
Effect of microwave radiation on the permeability of carbonic anhydrase loaded unilamellar liposomes; Orlando AR et al.; The influence of 2.45 GHz microwave exposure (6 mW/g) on the diffusion processes in enzyme-loaded unilamellar liposomes as bioreactors was studied . The enzyme carbonic anhydrase (CA) was entrapped into cationic unilamellar vesicles . Previous kinetic experiments showed a very low self-diffusion rate of the substrate p-nitrophenyl acetate (PNPA) across intact liposome bilayer . A twofold increase in the diffusion rate of PNPA through the lipid bilayer was observed after 120 min of microwave radiation compared to temperature control samples . The microwave effect was time dependent . The enzyme activity, as a function of increased diffusion of PNPA, rises over 120 min from 22.3% to 80% . The increase in stearylamine concentration reduces the enzyme activity from 80% to 65% at 120 min . No enzyme leakage was observed.

Physiol Res, 1994, 43(2), 137 - 9
Adrenergic lipolysis in immobilized and perfused adipocytes; Lincova D et al.; The method of cellular immobilization and perfusion was applied to adipocytes . The lipolytic effect of isoprenaline, whose action is produced as a result of receptor-drug interaction, was followed . An agarose solution kept at at 37 degrees C was mixed 1:1 with the cell suspension . Thereafter, adipocytes were immobilized in the agarose threads . The lipolytic effect of 0.1 ml of isoprenaline (1 x 10(-4) mol/l), that was rapidly introduced to the cell perfusion inlet in a non-recirculating system, was monitored by assessing glycerol production . The immobilized and perfused adipocytes exhibited significant lipolytic activity . After reaching the maximum effect, 0.1 ml of propranolol (1 x 10(-3) mol/l) that was applied to the bioreactor inlet, abolished the isoprenaline effect . The present data demonstrate the potential applicability of immobilized perfused adipocytes for various kinds of studies.

Physiol Res, 1994, 43(2), 131 - 5
A preliminary evaluation of drug biotransformation in hepatocytes of genetically defined rat strains; Hynie S et al.; This study was directed to use the genetically developed isoprenaline-sensitive (S), isoprenaline-resistant (R) and spontaneous hypertensive rats (SHR) as standard diseased animal models for in vitro liver function evaluation of drug biotransformation . Hepatic hexobarbital hydroxylase and glutathione transferase (GST) were evaluated by using hexobarbital and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates, at concentrations of 0.21 mmol/l and 1 mmol/l, respectively . The assay was conducted by using isolated hepatocytes in suspension and hepatocytes in a bioreactor configuration . The data demonstrate that there are certain cellular pharmacokinetic differences in hexobarbital hydroxylase and GST activities in hepatocytes obtained from Wistar, SHR, R and S strains which can be better demonstrated, when using the model of perfused and immobilized hepatocytes.

Physiol Res, 1994, 43(2), 127 - 30
Application of the hepatocytes bioreactor to xenobiotic biotransformation; Kamenikova L et al.; This study deals with the application of the previously developed immobilized and perfused isolated hepatocytes as a cellular system for the study of representative phase I and phase II of biotransformation reactions . To illustrate phase I reactions, aminopyrine (0.17-4.25 mmol/l) and hexobarbital (0.2 mmol/l) were selected . For phase II reactions, glutathione transferase activity was evaluated by using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate (0.125-2.0 mmol/l) . Formaldehyde, that was formed from aminopyrine, increased steadily in the perfusion medium with time . The perfused hepatocytes eliminated hexobarbital at a much higher rate than the hepatocytes in suspension . At several time points the amount of CDNB-glutathione conjugate formed per one million hepatocytes in the bioreactor was almost twice the amount formed by the hepatocytes in suspension . The present data illustrate the successful application of the hepatocyte bioreactor in phase I and phase II of xenobiotic metabolism and indicate that the cells were metabolically more active than the cells in suspension.

Physiol Res, 1994, 43(2), 121 - 5
Preparation of functionally active immobilized and perfused mammalian cells: an example of the hepatocyte bioreactor; Farghali H et al.; In the present study, a method has been employed for hepatocyte immobilization in agarose threads which allows for cell perfusion . The rat hepatocytes are isolated from the liver . A 1.8% low-gelling agarose solution is prepared in warm Krebs-Henseleit solution . The agarose solution is mixed 1:1 with the hepatocytes and the cells are immobilized in agarose threads by extruding the agarose-cell mixture through cooled Chemfluor teflon (TFE) tubing . Light and electron microscopy studies indicated the integrity of the hepatocytes in the gel matrix . This system allows for liver cell perfusion and viability studies to be carried out non-invasively on the cells and provides data that are comparable to those obtained with a perfused isolated liver . Immobilized hepatocytes are an in vitro system worthy of further evaluation which may be useful in the studies of liver cell metabolism and the response of the liver to foreign chemicals.

Physiol Res, 1994, 43(2), 117 - 20
The concept of application of immobilized and perfused mammalian cells (a bioreactor model) in biomedical research; Farghali H et al.; An overview of the concept of cellular immobilization and perfusion as a small laboratory bioreactor model is presented . The cellular systems currently used may be described as static . This is due to conditions of hypoxia and waste product build-up that affect cell physiology . Cellular immobilization and perfusion is, therefore, expected to maintain the cells for very long periods of time under approximately physiological conditions . A number of applications of immobilized perfused hepatocytes and other cellular systems such as adipocytes and Sertoli cells are described in addition to various other cell lines . Moreover, it is suggested that the bioreactor may have potential use as a bioartificial organ.

Biomed Pharmacother, 1994, 48(7), 282 - 6
Endosome-lysosomes and neurodegeneration; Mayer RJ et al.; A number of the major human and animal neurodegenerative diseases, such as Alzheimer's disease and sheep scrapie, are characterised by deposits of amyloid, arising through incomplete breakdown of membrane proteins . Although our knowledge concerning these diseases is increasing, they remain largely untreatable . Recently, attention has focussed on the mechanisms of production of different types of amyloid and the likely involvement within cells of acid compartments called endosome-lysosomes . These organelles may be 'bioreactor' sites for the unfolding and partial degradation of membrane proteins to generate the amyloid materials . These subsequently become expelled from the cell, or are released from dead cells, and accumulate as pathological entities . Common features of the disease processes give new direction to therapeutic intervention.

J Hematother, 1994 Fall, 3(3), 213 - 8
Future paradigm for autologous bone marrow transplantation: tumor purging and ex vivo production of normal stem and progenitor cells; Rummel SA et al.; A major concern in autologous bone marrow transplantation (ABMT) is the possible contamination of the graft with tumor cells . Transplantation of malignant cells, along with normal hematopoietic stem and progenitor cells, may contribute to relapse of disease . Therefore, a growing strategy is to subject autologous marrow to some type of purging procedure to eliminate tumor cells selectively . Transplantation of purged marrow, however, often results in a delayed engraftment associated with (specific or nonspecific) loss of normal stem and progenitor cells during manipulations related to the purging process . A new and burgeoning field in the area of clinical bone marrow transplantation is the ex vivo production of stem and progenitor cells . Several advantages accrue to this strategy . First, this technology makes it possible to expand the stem and progenitor cell population of a small volume of bone marrow or mobilized peripheral blood (MPB), thus lessening the initial tumor burden to be purged . Secondly, ex vivo marrow or MPB expansion may overcome the significant problem of delayed engraftment by rebuilding the numbers of normal stem and progenitor cells necessary for both early and durable engraftment . To accomplish these and other objectives, an automated and closed, clinical-scale bioreactor system, based on continuous perfusion technology, is being developed and will soon enter clinical trials.

Cancer Biother, 1994 Fall, 9(3), 211 - 24
Growth of tumor derived activated T-cells for the treatment of cancer; Lewko WM et al.; This report describes the production of Tumor Derived Activated Cell cultures (TDAC, also called tumor infiltrating lymphocytes) from patient tumor biopsies and our preliminary experience growing these cells to therapeutic levels using artificial capillary bioreactor cultures . TDAC were successfully grown in medium containing Interleukin 2 from 80% of the 113 tumor biopsies tested . There was no significant difference in success (growth to 1 x 10(9) cells) comparing primary and metastatic tumors . Many of the tumors were shipped to the laboratory from distant sites . Success rate did decrease with the length of time for tumor transport . Interleukin 4 was beneficial in the development of 1 of 4 TDAC cultures which did not grow with IL-2 only . Seventy-seven bioreactor cultures were initiated for 31 patients . On the average, 1.9 x 10(9) TDAC were inoculated per bioreactor; 3.3 x 10(10) were harvested in 22 days . Twelve liters of medium were required per 1 x 10(10) TDAC produced . TDAC cultures contained T cells with variable ratios of CD4 to CD8 cells . Secreted granulocyte monocyte colony stimulating factor, interferon gamma and tumor necrosis factor were measured in the bioreactor cartridge conditioned medium . Twenty three patients were evaluated . Partial responses were observed in 4 patients including a dramatic remission of scalp nodules in a patient with renal cancer . Results showed that therapeutic amounts of TDAC cells may be produced in a reasonable and cost effective manner using artificial capillary bioreactor cultures.

Cytotechnology, 1994, 15(1-3), 351 - 63
Towards the development of a bioartificial pancreas: immunoisolation and NMR monitoring of mouse insulinomas; Sambanis A et al.; A promising method for diabetes treatment is the implantation of immunoisolated cells secreting insulin in response to glucose . Cell availability limits the application of this approach at a medically-relevant scale . We explore the use of transformed cells that can be grown to large homogeneous populations in developing artificial pancreatic tissues . We also investigate the use of NMR in evaluating, non-invasively, cellular bioenergetics in the tissue environment . The system employed in this study consisted of mouse insulinoma beta TC3 cells entrapped in calcium alginate/poly-L-lysine (PPL)/alginate beads . The PPL layer imposed a molecular weight cutoff of approximately 60 kDa, allowing nutrients and insulin to diffuse through but excluding high molecular weight antibodies and cytotoxic cells of the host . We fabricated a radiofrequency coil that can be double-tuned to 1H and 31P, and an NMR-compatible perfusion bioreactor and support circuit that can maintain cells viable during prolonged studies . The bioreactor operated differentially, was macroscopically homogeneous and allowed the acquisitions of 1H images and 31P NMR spectra in reasonable time intervals . Results indicated that entrapment had little effect on cell viability; that insulin secretion from beads was responsive to glucose; and that the bioenergetics of perfused, entrapped cells were not grossly different from those of cells never subjected to the immobilization procedure . These findings offer promise for developing an artificial pancreatic tissue for diabetes treatment based on continuous cell lines.

Cytotechnology, 1994, 15(1-3), 321 - 8
Quantitative investigations of cell-bubble interactions using a foam fractionation technique; Tan WS et al.; Previous work by the authors and others has shown that suspended animal cell damage in bioreactors is caused by cell-bubble interactions, regardless whether the bubbles are from bubble entrainment or direct gas sparging . As approach to measure the adsorptivity of animal cells to bubbles, a modified batch foam fractionation technique has been developed in this work and proven to be applicable . By using this technique, the number of cells absorbed per unit bubble surface area and the adsorption coefficients have been measured to quantify hybridoma cell-bubble interactions, and the preventive effects of serum and Pluronic F68 on these interactions . It was demonstrated quantitatively that the hybridoma cells adhere to bubbles spontaneously and significant numbers exist in the foam, and that both the serum and Pluronic F68 provide strong prevention to these cell-bubble interactions . The results obtained provide criteria for bioreactor operation and medium formulation to prevent cell-bubble interactions and cell damage in the culture processes.

Cytotechnology, 1994, 15(1-3), 311 - 20
Cells and bubbles in sparged bioreactors; Chalmers JJ; Ever since animal cells have been grown in-vitro, various techniques have been used to supply the cells with oxygen . The most simple and commonly used 'large-scale' technique to provide oxygen is through the introduction of gas bubbles . However, almost since the beginning of in-vitro cell culture, empirical observations have indicated that bubbles can be detrimental to the cells . This review will discuss the background of the problem, review the relevant research on the topic, attempt to provide a coherent summary of what we know from all of this research, and finally outline what still needs to be investigated . Specific topics to be covered include: experimental correlations of cell damage with bubbles, cell attachment to bubbles, the hydrodynamics of bubble rupture, bioreactor studies, visualization studies, and computer simulations and qualification of cell death as a result of bubble rupture.

Cytotechnology, 1994, 15(1-3), 301 - 9
Design of a bubble-swarm bioreactor for animal cell culture; Gudermann F et al.; A stationary bubble-swarm has been used to aerate a mammalian cell culture bioreactor with an extremely low gas flow rate . Prolonging the residence time of the gas bubbles within the medium improved the efficiency of the gas transfer into the liquid phase and suppressed foam formation . An appropriate field of speed gradients prevented the bubbles from rising to the surface . This aeration method achieves an almost 90% transfer of oxygen supplied by the bubbles . Consequently, it is able to supply cells with oxygen even at high cell densities, while sparging with a gas flow of only 0.22 x 10(-3) -1.45 x 10(-3) vvm (30-200 ml/h) . The reactor design, the oxygen transfer rates and the high efficiency of the system are presented . Two repeated batch cultures of a rat-mouse hybridoma cell line are compared with a surface-aerated spinner culture . The used cell culture medium was serum-free, either with or without BSA and did not contain surfactants or other cell protecting agents . One batch is discussed in detail for oxygen supply, amino acid consumption and specific antibody production.

Cytotechnology, 1994, 15(1-3), 3 - 9
Cultural and physiological factors affecting expression of recombinant proteins; Griffiths JB et al.; The variability in expression of recombinant proteins has been analyzed with regard to (a) comparison of clones from the same transfection experiment; (b) comparison of the same genetic construct in different cell lines; (c) the effect of the culture system used (free suspension, aggregate suspension, and microcarrier); and (d) physiochemical parameters in long-term (100d) culture in a macroporous fixed bed bioreactor (FBR) . Differences in product expression between clones were accompanied by differences in growth rates, metabolic kinetics, and ability to grow in suspension as opposed to attached culture . The single most important factor affecting product expression when comparing constructs (for SEAP and IgG), cell lines (BHK 21 and myeloma), and culture systems was whether cells were grown in an attached or suspension mode . Thus key factors could be related to cell morphology (suspension versus monolayer), the presence of microenvironments and physiological stress to control growth rate . The relationship of key process parameters to volumetric and specific rAb productivity of the FBR was investigated in a partial factorial experiment with a rBHK cell line . The highest productivity levels are associated with a combination of the highest values tested for re-cycle (195 ml min-1) and dilution rates (1 d-1) and glutamine concentration (2.5 mmol 1-1), plus the lowest values for bead size (2 mm) and inoculum density (10(7) m1-1) . Together with data from fluidised bed cultures, these results suggest that higher productivity is not primarily the result of greater cell numbers within the system but more the physicochemical definition of the system.

Cytotechnology, 1994, 15(1-3), 271 - 9
A direct computer control concept for mammalian cell fermentation processes; Buntemeyer H et al.; In the last 10 years, new assignments and the special demands of mammalian cells to the culture conditions caused the development of complex small scale fermentation setups . The use of continuous fermentation and cell retention devices requires appropriate process control systems . An arrangement for control and data-acquisition of complex laboratory-scale bioreactors is presented . The fundamental idea was the usage of a standard personal computer, which is connected to pumps, valves and sensors via ADA-transformation . The possibility of free programming allowed the development of user-oriented software, especially designed for the far-reaching requirements of a university laboratory in the field of animal cell culture . Control of aeration, pumps, data-acquisition and data-storage are combined within one program, which allows the automation of standard operations like measurement of kLa- or OTR-values . Pump control algorithms for all common fermentation strategies (batch, fed batch, chemostat, perfusion) are included and can be selected any time during cultivation . Oxygen partial pressure and pH are controlled via direct digital control (ddc), providing simple adaption of control parameters and set points to current fermentation conditions.

Cytotechnology, 1994, 15(1-3), 253 - 8
Vortex flow filtration of mammalian and insect cells; Hawrylik SJ et al.; The use of vortex flow filtration for harvesting cells or conditioned medium from large scale bioreactors has proven to be an efficient, low shear method of cell concentration and conditioned medium clarification . Several 8-10 L batches of the human histiocytic lymphoma U-937 cell line (ATCC CRL 1593) were concentrated to less than 1 L by vortex flow filtration through a 3.0 microns membrane . An aggressive filtration regimen caused a 17% loss of cell viability and a 32% loss of IL-4 receptor binding capacity when compared to a batch centrifuged control . A reduction of the rotor speed from 1500 to 500 RPM and reduction of system back pressure from 10 to 0 PSIG resulted in cell viability and IL-4 binding capacity comparable to the control . Several 10 L batches of baculovirus infected Sf-9 cells were also concentrated to less than 1 L by vortex flow filtration through a 3.0 microns membrane . SDS-PAGE analysis of filtrate samples showed that aggressive filtration caused cell damage which led to contamination of the process stream by cellular lysate . When rotor speed was reduced to 500 RPM and system back pressure was reduced to 0 PSIG, the amount of contaminating lysate proteins in filtrate samples was comparable to a batch centrifuged control.

Cytotechnology, 1994, 15(1-3), 177 - 86
Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells; Hensler WT et al.; The baculovirus infection process of Spodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinant Autographa californica (AcNPV) virus expressing beta-galactosidase enzyme as a model system . A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection . The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident . Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection . The optimal harvest time varied, depending on which technique--SDS-PAGE, chromogenic (ONPG) or fluorometric (C12FDG)--was used to monitor beta-galactosidase production . Specific beta-galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units beta-galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.

Cytotechnology, 1994, 15(1-3), 17 - 29
Applications of improved stoichiometric model in medium design and fed-batch cultivation of animal cells in bioreactor; Xie L et al.; In our previous work (Xie and Wang, 1994a), a simplified stoichiometric model on energy metabolism for animal cell cultivation was developed . Fed-batch experiments were performed in T-flasks using this model in supplemental medium design (Xie and Wang, 1994b) . In this work, the major pathways of glucose and glutamine metabolism were incorporated into the stoichiometric model . Fed-batch culture was conducted in a 2-liter bioreactor with appropriate process control strategies . Nutrient concentrations, especially glucose and glutamine, were maintained at constant but low levels through the automated feeding of a supplemental medium formulated using the improved stoichiometric model . The formation of toxic byproducts, such as ammonia and lactate (Hassell et al., 1991), was greatly reduced . The specific lactate production rate was decreased by 62-fold compared with batch culture in bioreactor and by 8-fold compared to fed-batch culture in T-flask using the previous stoichiometric model . Ammonia formation was also decreased compared with both the batch and fed-batch cultures . Most importantly, the monoclonal antibody concentration reached 900 mg l-1, an increase of 17- and 1.6-fold compared with the batch and fed-batch cultures respectively.

Cytotechnology, 1994, 15(1-3), 157 - 67
Relationship between oxygen uptake rate and time of infection of Sf9 insect cells infected with a recombinant baculovirus; Wong TK et al.; Oxygen uptake rates (OUR) of Sf9 insect cells propagated in a serum-free medium (SF900II, Gibco) and of cells infected with a recombinant AcNPV were investigated before and after infection in a laboratory-scale bioreactor . The volumetric OURs of uninfected and exponentially growing cells were found to be proportional to the cell density . For infected cultures, the specific OUR of cells increased immediately after addition of virus and a maximum of 1.3 times the value of uninfected cells was noted for all the cultures between 8 to 30 hours post infection, which coincides with the period at which most viral replication and the majority of DNA synthesis takes place . It was observed that the rate of rise in the specific OUR decreased as the cell density at the time of infection increased, which meant that the later the infection, the later the maximum sOUR was observed . We therefore suggest that OUR measurement can be used to reflect the efficiency of a batch infection . Carbohydrate and amino acid consumption rates from an infected run were analysed in an effort to identify substrate(s) that may be used at increased rates to fuel the rise in oxygen demand observed early in the infection cycle . No observable rise in the consumption rates of glucose or glutamine, which are the major energy sources for animal cells, were seen after infection but an increase in the consumption rates of some amino acids suggests that infected Sf9 cells may utilise amino acids at an enhanced rate for energy post infection.

Cytotechnology, 1994, 15(1-3), 145 - 55
Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells; Garnier A et al.; Human 293S cells, a cell line adapted to suspension culture, were grown to 5 x 10(6) cells/mL in batch with calcium-free DMEM . These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content . Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5 x 10(5) cells/mL . In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C, how product yield can be maintained at high cell densities of 2 x 10(6) cells/mL by a medium replacement strategy . This allows the production of as much as 90 mg/L of active recombinant protein per culture volume . Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM . Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system . The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.

Cytotechnology, 1994, 15(1-3), 117 - 28
Induction of apoptosis in oxygen-deprived cultures of hybridoma cells; Mercille S et al.; It is now well documented that apoptosis represents the prevalent mode of cell death in hybridoma cultures . Apoptotic or programmed cell death occurs spontaneously in late exponential phase of batch cultures . Until lately, no specific triggering factors had been identified . Recently, we observed that glutamine, cystine or glucose deprivation induced apoptosis in both hybridoma and myeloma cell lines whereas accumulation of toxic metabolites induced necrotic cell death in these cells . Other triggering factors such as oxygen deprivation might also be responsible for induction of apoptosis . In the present study, induction of cell death by exposure to anoxia was examined in batch culture of the SP2/0-derived hybridoma D5 clone . The mode of cell death was studied by morphological examination of acridine orange-ethidium bromide stained cells in a 1.5 L bioreactor culture grown under anoxic conditions for 75 hours . Under such conditions, viable cell density levelled off rapidly and remained constant for 25 hours . After 45 hours of anoxia, cell viability had decreased to 30% and the dead cell population was found to be 90% apoptotic . In terms of cellular metabolism, anoxia resulted in an increase in the utilization rates of glucose and arginine, and in a decrease in the utilization rate of glutamine . The lactate production rate and the yield of lactate on glucose increased significantly while the MAb production rate decreased . These results demonstrate that glycolysis becomes the main source of energy under anoxic conditions . Cells incubated for 10 hours or less under anoxic conditions were able to recuperate almost immediately and displayed normal growth rates when reincubated in oxic conditions whereas cells incubated for 22 hours or more displayed reduced growth rates . Nonetheless, even after 22 h or 29 h of anoxia, cells reincubated in oxic conditions showed no further progression into apoptosis . Therefore, upon removal of the triggering signal, induction of apoptosis ceased.

Cytotechnology, 1994, 15(1-3), 111 - 6
Low temperature cultivation--a step towards process optimisation; Weidemann R et al.; Adherent recombinant BHK cells were cultivated at temperatures between 30 and 37 degrees C . Batch and repeated-batch-cultivations in a 2-litre bioreactor showed a significant influence on metabolism and cell growth . The low-temperature-cultivations showed a lower growth rate and a lower glucose consumption rate and, therefore, less lactate production . On the other hand, the maximum cell density and productivity seemed not to be affected by the temperature reduction.

Cytotechnology, 1994, 16(1), 51 - 8
Hybridoma cells in a protein-free medium within a composite gel perfusion bioreactor; Shen BQ et al.; A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels . The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper . During the continuous operation of this system, the total cell density reached 3.9 x 10(7) cells per ml of beads (viability 79.6%) . The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 micrograms per 10(6) viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium . Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed . Leaching of materials from the beads was evident and the major fraction of released materials was alginate.

Cytotechnology, 1994, 14(3), 219 - 32
Methods and strategies available for the process control and optimization of monoclonal antibody production; Fu P et al.; The objective of this paper is to explore the range of methods and strategies available for the process control and optimization of monoclonal antibody production by hybridoma cell culture . Emphasis will be placed on the choice of the level of complexity incorporated into the process control and optimisation procedure . It will be shown that the behaviour of hybridomas in culture is influenced by sophisticated cellular metabolic activities and various interactive environmental factors and that the understanding and modelling of the way hybridomas grow in the bioreactor should enable optimisation of bioreactor operating conditions to achieve maximum monoclonal antibody formation . However, due to the lack of on-line instrumentation of important biological variables and the incomplete knowledge of hybridoma cultivation process, there exist many limitations and challenges to the advent of applications of process control and optimisation in this field . To solve the problem, introduction of industrially practical biological measurements and development of new control concepts are inevitable . At the end of this paper, we shall discuss possible schemes for the control of the physiological state of cells in order that balanced cell growth and maximum monoclonal antibody synthesis may be achieved.

Cytotechnology, 1994, 14(3), 183 - 90
High density culture of mammalian cells with dynamic perfusion based on on-line oxygen uptake rate measurements; Kyung YS et al.; In a continuous culture with cell retention the perfusion rate must be adjusted dynamically to meet the cellular demand . An automated mechanism of adjusting the perfusion rate based on real-time measurement of the metabolic load of the bioreactor is important in achieving a high cell concentration and maintaining high viability . We employed oxygen uptake rate (OUR) measurement as an on-line metabolic indicator of the physiological state of the cells in the bioreactor and adjusted the perfusion rate accordingly . Using an internal hollow fiber microfiltration system for total cell retention, a cell concentration of almost 10(8) cells/mL was achieved . Although some aggregates were formed during the cultivation, the viability remained high as examined with confocal microscopy after fluorescent vital staining . The results demonstrate that on-line OUR measurement facilitates automated dynamic perfusion and allows a high cell concentration to be achieved.

Cytotechnology, 1994, 14(3), 167 - 75
Use of on-line gas analysis to monitor recombinant mammalian cell cultures; Lovrecz G et al.; The development of a system capable of accurately measuring the oxygen uptake and carbon dioxide production rates during mammalian cell cultures is described . A detailed study of the specifications of the various components used in the system for the measurement of gas flow rates and composition, coupled with the validation of the system independent of the bioreactor was carried out . The aim of this study was to identify and eliminate where possible the errors controlling the accuracy of determination of gaseous metabolic rates . This study showed the importance of controlling the temperature of gaseous oxygen entering the system . With such temperature control, it was possible to obtain data with an accuracy of +/- 5% at the 95% confidence level . Another source of error, the use of bi-carbonate buffer, was studied . A mathematical model was used to compensate for the affect of such buffers on the determination of carbon dioxide production rates . The use of the system for the continuous determination of gaseous metabolism during the growth and production phase for recombinant CHO cell cultures is described.

Cytotechnology, 1994, 14(3), 157 - 65
Mass spectrometry: a tool for on-line monitoring of animal cell cultures; Behrendt U et al.; The magnetic sector mass spectrometer is able to detect oxygen uptake and carbon dioxide production rates from animal cell cultivations performed in 101 bioreactors . Such data have not been available with the use of classic exhaust gas analysis applying paramagnetic analyzers and infra-red sensors due to the insensitivity of the apparatus available . In the course of the present work we were able to demonstrate, that the oxygen uptake rate correlates to the number of viable cells . Additionally oxygen uptake rates supplied on-line information about the actual physiology of the cells: When the rates changed during the cultivation process, this immediately indicated the occurrence of limitations of components in the medium . The information could be useful in timing key events, such as performing splits or harvesting the bioreactor.

Cytotechnology, 1994, 14(1), 67 - 80
Bead-to-bead transfer of Chinese hamster ovary cells using macroporous microcarriers; Ohlson S et al.; Chinese hamster ovary cells (CHO-K1) were cultivated in macroporous gelatin microcarriers (CultiSpher G and CultiSpher S) in spinner flasks and a 51 bioreactor . Near-to-confluent cultures were harvested by bead-to-bead transfer where intact microcarriers with cells were transferred from a spinner flask to another spinner flask or to the bioreactor with naked microcarrier beads . Successful bead-to-bead transfer was achieved in various split ratios . The duration of attachment seemed to be important where the direct contact of beads to each other can be achieved by intermittent stirring . Repeated transfers were performed and at least four transfers in spinner flasks were achieved . Two variations of bead-to-bead transfer were performed in the 51 bioreactor either by seeding the bioreactor with near-to-confluent beads cultivated in spinner flasks or in situ transfer by adding fresh beads to the bioreactor . As in the spinner case, attachment was achieved by intermittent stirring where donor beads were in close proximity to the acceptor beads . Again successful transfers were obtained as evidenced by the good growth on acceptor beads where cell yields were in the range of 3100-4500 cells/bead . The results suggest that bead-to-bead transfer of CHO-K1 cells can be easily performed and do provide an alternative route to applications where dissolution techniques may not offer an efficient solution.

Cytotechnology, 1994, 14(1), 61 - 6
High-density culture of FM-3A cells using a bioreactor with an external tangential-flow filtration device; Kawahara H et al.; A novel bioreactor system developed for high-density cultures of suspended mammalian cells is described using a tangential-flow filtration device outside the culture vessel to separate viable cells from spent medium . The filtration device is based on thin porous microfiltration membranes with a pore size of 0.20-0.65 microns . Because cells have a diameter of about 10-20 microns, they cannot permeate these membranes with the spent medium . So, allowing a perfusion culture to be created using this system . In most membrane filtration systems, clogging of the membranes has made long-term operation difficult . In this system, however, high pressure is not applied directly to the membrane, thus minimizing clogging . Also, clogging of the membrane was prevented by washing the membrane surface once a day, and increasing the membrane surface area . With this system, FM-3A cells were cultured and maintained at a high density of 3.0 x 10(7) cells/ml for two weeks, and a continuous culture was supported for as long as 34 days.

Cytotechnology, 1994, 14(1), 1 - 9
The Super-Spinner: a low cost animal cell culture bioreactor for the CO2 incubator; Heidemann R et al.; The production of small quantities of monoclonal antibodies and recombinant proteins was carried out using a new low cost production system, the Super Spinner . Into a 1 1 standard Duran flask a membrane stirrer equipped with a polypropylene hollow fiber membrane was installed to improve the oxygen supply by bubble-free aeration . The aeration was facilitated by using the CO2 conditioned incubator gas, which was pumped through the membrane stirrer via a small membrane pump . The maximal oxygen transfer rate (OTRmax) of the Super Spinner was detected . For this purpose one spinner flask was equipped with an oxygen electrode . The OTRmax was measured by the dynamic method . The ratio of membrane length to culture volume was adapted corresponding to the oxygen uptake rate of the cells according to the desired cell density . A balanced nutrient supply resulted in an optimal formation and yield of products.

Biotechnol Prog, 1994 Jan-Feb, 10(1), 60 - 4
Continuous beta-galactosidase production in insect cells with a p10 gene based baculovirus vector in a two-stage bioreactor system; van Lier FL et al.; Continuous production of polyhedra or baculovirus-expressed proteins in insect cell cultures is limited to about 4 weeks . The decrease in production has been ascribed to the interference of defective deletion mutants with wild-type baculoviruses . The deleted genome sequences include the polyhedrin gene (or the heterologous gene of interest); in the remaining part, the major late p10 gene is always maintained . In the present study, the productivity of a recombinant baculovirus with the lacZ gene from Escherichia coli cloned downstream of the p10 promoter at the p10 locus was investigated . It was hypothesized that this p10 promoter driven gene is preserved over a longer period of time in a continuously operated two-stage bioreactor system than foreign genes behind the polyhedrin promoter at the polyhedrin locus . In two separate runs, beta-galactosidase production with the p10-lacZ recombinant reached quasi-steady-state levels of 30 and 60 units/cm3 . Polyhedron production was about 3 x 10(6) and 6 x 10(6) polyhedra/cm3, respectively . However, both polyhedron and beta-galactosidase production decreased after about 30 days of relatively constant production . In the infection reactor, deletion mutants of the virus, which contained both the polyhedrin and lacZ gene, were predominant . Therefore, the presence of the polyhedrin or p10 gene alone in deletion mutants is not sufficient for prolonged expression; other genes involved in major late gene expression and not present in the deleted virus are probably necessary.

Biotechnology (N Y), 1994 Jan, 12(1), 75 - 8
Measurements of the growth and distribution of mammalian cells in a hollow-fiber bioreactor using nuclear magnetic resonance imaging; Callies R et al.; We have used diffusion-weighted 1H NMR micro-imaging and localized spectroscopy techniques to monitor the growth and distribution of mammalian cells in a hollow-fiber bioreactor . Non-invasive NMR measurements of this type should also allow investigation of metabolic heterogeneity and assist in future designs of hollow-fiber systems.

Hum Antibodies Hybridomas, 1994, 5(3-4), 98 - 104
Antibody production of a human EBV-transformed B cell line and its heterohybridoma and trioma cell line descendants in different culture systems; Gustafsson B et al.; Cells of an EBV-transformed human lymphoblastoid B cell line, producing antibodies directed against tetanus toxin, were fused with mouse myeloma cells (SP2/0) and with mouse-human heteromyeloma cells (SPAM-8) resulting in the formation of heterohybridoma and trioma cells, respectively . Antibody production of the three cell lines were studied under different culture conditions . All three cell lines produced antibodies in concentrations ranging from 2.6 to 6.4 micrograms ml-1 in spent medium from stationary flask cultures . Dialysis cultures of trioma and heterohybridoma cells resulted in concentrations of 36 and 20 micrograms ml-1, respectively, whereas no significant increase was obtained with the EBV-transformed cells . Trioma cells, cultured in a hollow fiber cartridge bioreactor produced antibodies in concentrations of average of 303 micrograms ml-1, whereas the EBV-transformed cells did not adapt to this system . Furthermore, trioma and heterohybridoma cells injected into the intraperitoneal cavity of SCID-mice, produced antibodies in ascites fluid in concentrations of 500 and 640 micrograms ml-1 respectively.

Hum Antibodies Hybridomas, 1994, 5(3-4), 143 - 51
Galactosylation of human IgG monoclonal anti-D produced by EBV-transformed B-lymphoblastoid cell lines is dependent on culture method and affects Fc receptor-mediated functional activity; Kumpel BM et al.; Human monoclonal antibodies to the Rh D blood group antigen were produced by EBV-transformed B cell lines grown in serum-free medium in low density (LD) static cultures or high density (HD) hollow fiber bioreactors . Glycosylation analysis of the purified IgG was determined by the binding of anti-GlcNAc monoclonal antibody (GN7) and by analysis of oligosaccharides released by hydrazinolysis . The LD MAbs had only trace levels of agalactosyl oligosaccharides (G0), the major species (> 70%) being digalactosyl structures (G2) . The HD MAbs, by contrast, contained about 10% G0 and relatively high levels (over 50%) of monogalactosyl (G1) oligosaccharides . beta 1-4 galactosyltransferase activity in the LD cell lines was similar to that found previously for other EBV-transformed B cell lines . The predominant oligosaccharides of an IgG3 anti-D, BRAD-3, contained bisecting N-acetylglucosamine . In functional assays with Fc receptor (Fc gamma R) positive effector cells, the highly galactosylated LD form of BRAD-3 was more active than the HD form in monocyte (Fc gamma RI) and K cell (Fc gamma RIII) mediated lysis of erythrocytes in ADCC assays, although these preparations showed no difference in Fc gamma RI-mediated rosette formation with U937 cells . One MAb, JAC10, was over 10-fold less active than two other IgG1 MAbs, 2B6 and BRAD-5, at mediating lysis of erythrocytes by Fc gamma RIII+ K cells; differences in sialylation may have contributed to this heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adv Exp Med Biol, 1994, 368, 165 - 71
Use of hepatocyte cultures for liver support bioreactors; Gerlach JC; Hybrid artificial liver systems are being developed as extracorporeal temporary liver support therapy . Here, an overview is given with emphasis on hepatocyte culture models for bioreactors, in vitro studies, animal studies and the clinical application of hybrid liver support systems . In vitro studies show long term external metabolic functions of primary isolated hepatocytes in bioreactors . These systems are capable of supporting essential liver functions . Animal experiments show the possibility of upscaling the bioreactors for clinical treatment . Since there is no reliable animal model for investigations on the treatment of acute liver failure, the promising results of these studies have limited relevance . The small number of clinical studies are not sufficient to give statements about a clinical improvement of therapy of acute liver failure . Although important progress has been made in the development of the systems, multiple different hepatocyte culture models and bioreactor constructions are discussed in the literature, indicating competition in this field of medical research.

Blood Cells, 1994, 20(2-3), 482 - 90; discussion 491
Expansion in bioreactors of human progenitor populations from cord blood and mobilized peripheral blood; Van Zant G et al.; Umbilical cord blood (UCB) and mobilized peripheral blood (MPB) provide an alternate source to bone marrow for transplantation . Expansion in vitro of stem/progenitor cell populations from these sources may provide adult-sized grafts otherwise not attainable because of the limited cell numbers available in the case of UCB or because of numerous rounds of apheresis required for sufficient MPB cells . We asked whether continuous perfusion culture could be employed in ex vivo expansion to produce clinically relevant numbers of stem/progenitor cells from these sources . To evaluate MPB, 1-10 million leukocytes, from patients who had received either granulocyte colony-stimulating factor (G-CSF) or cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF), were inoculated into bioreactors, with or without irradiated, allogeneic stroma . The growth factor combination in the perfusion medium consisted of interleukin-3 (IL-3), stem cell factor (SCF), GM-CSF and erythropoietin (Epo) . Under the best conditions tested, total cell numbers, granulocyte-macrophage colony-forming units (CFU-GM), and long-term culture-initiating cell (LTC-IC) populations were expanded by about 50-, 80-, and 20-fold, respectively, over 14 days . At low cell inocula (1 million), the presence of stroma enhanced the expansion of total cells and CFU-GM but not of LTC-IC . When SCF was not included in the medium, both total cells and CFU-GM expanded to a much lesser extent, but again the expansion of LTC-IC was not affected . At the higher cell inoculum (10 million), expansions of total cells and CFU-GM were equivalent with or without stroma . To evaluate UCB, cells were placed into bioreactors with or without irradiated, allogeneic stroma, and the bioreactors were perfused with medium containing the four standard growth factors . After 6-14 days, in several independent experiments, 20-24 million cells were harvested from bioreactors perfused with SCF-containing medium, irrespective of the presence or absence of preformed stroma . Similarly, in reactors perfused with SCF-containing medium (with or without stroma), an average 40- to 60-fold expansion of CFU-GM was obtained, yielding an average of 1.5-1.8 x 10(5) CFU-GM per reactor . Harvested cells were thus up to 40-fold enriched in CFU-GM in comparison to the inoculum . In the absence of SCF, cell expansions averaged 1.5- to 2-fold, and CFU-GM were expanded only 10- to 14-fold by day 14 . As before, the presence of preformed stroma did not affect either cell or CFU-GM yields, provided the cell inoculum was at least 4.5 million cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Eur J Cancer, 1994, 30A Suppl 3, S2 - 6
Applications of recombinant DNA technology in the production of glycosylated recombinant human granulocyte colony stimulating factor; Holloway CJ; Lenograstim has been developed by recombinant DNA technology and is expressed in large-scale mammalian cell culture . It has been shown that lenograstim is indistinguishable in its physicochemical, structural and biological properties with respect to native granulocyte colony stimulating factor isolated from a human cell line . In particular, both the recombinant and natural proteins have identical amino acid sequences, contain the same intra-polypeptide chain disulphide bridges and exhibit the same posttranslational carbohydrate structures . Lenograstim is manufactured by expanding inoculum from vials of the Manufacturer's Working Cell Bank (from molecular cloning) followed by culture in a large bioreactor . Purification of lenograstim involves a four-step chromatographic process . The active ingredient is monitored by in-process controls at all stages of manufacture and routinely as purified bulk . The finished product is formulated into excipients reflecting conditions close to the natural environment of the protein with respect to pH, osmolarity and the presence of human serum albumin.

Dev Biol Stand, 1994, 83, 55 - 64
Hybridoma stability; Castillo FJ et al.; Hybridoma stability issues include mutations, chromosome losses, and the potential effects of process variables on the yield, quality and homogeneity of the Monoclonal Antibody (MAb) product . MAb production by murine hybridomas is typically unstable in the early stages after fusion but repeated cloning normally produces stable clones . The stability of hybridomas and the consistency of the MAbs produced during extended high density perfusion cultures at Xoma Corporation were evaluated . Cell stability was assessed by recovering cells from the bioreactors at different intervals and comparing their growth and product formation kinetics and yields to those of cells started fresh from the corresponding Manufacturer's Working Cell Banks . Product consistency was evaluated in the crude harvests and in the corresponding purified MAb lots by biochemical and functionality tests including: SDS-PAGE (reducing and non-reducing), IEF, HPLC (size exclusion and cation exchange), peptide mapping, N-terminal sequencing, carbohydrate composition and binding assays . Several murine hybridomas were studied during runs lasting several months and found to be stable by all criteria employed . Such results support the viability of extended hollow fiber perfusion cultures for reproducible production of murine MAbs . Selecting stable clones and understanding the effects of process variables on the quantity and quality of the MAbs are keys to controlling hybridoma stability during the manufacturing process.

Gene, 1993 Dec 15, 135(1-2), 37 - 47
The origin of genetic information: viruses as models; Eigen M; A living entity can be described as a complex adaptive system which differs from any, however complex, chemical structure by its capability of functional self-organization based on the processing of information . If one asks, where does this information come from and what is its primary semantics, the answer is: information generates itself in feedback loops via replication and selection, the objective being 'to be or not to be' . This paper describes the theoretical framework of information-generating systems and provides experimental clues for some basic forms of genetic organization, such as molecular quasi-species, hypercyclic and compartmentalized RNA-protein assemblies . The results are primarily obtained with RNA viruses and virus-like systems . The experiments are carried out with the help of automated, computer-controlled bioreactors, called 'evolution machines', that may form the basis of a new 'evolutionary biotechnology'.

Biotechnol Appl Biochem, 1993 Dec, 18 ( Pt 3), 217 - 26
Methanol detoxification by enzyme-loaded erythrocytes; Magnani M et al.; Alcohol oxidase (AlOx) from Pichea pastoris (a methylotrophic yeast) was encapsulated into human and murine erythrocytes up to 2 units/ml of packed cells . This enzyme has a much higher affinity for methanol than for ethanol, thus making the loaded erythrocytes useful cellular bioreactors able to catabolize methanol . Enzyme-loaded erythrocytes showed an increased rate of the hexose-monophosphate-shunt activity and a significant methaemoglobin production . However, the in vivo survival of these cells does not seem to be significantly affected by methanol catabolism . In vivo, mice receiving AlOx-loaded erythrocytes were able to keep the blood methanol concentrations below values that were about 50% of those found in mice receiving unloaded cells and similar amounts of methanol . Thus AlOx-loaded erythrocytes may add an important contribution to the detoxification protocol against methanol poisoning.

Anal Chem, 1993 Dec 1, 65(23), 3363 - 7
Automated process monitoring of monoclonal antibody production; Paliwal SK et al.; Antifibronectin, monoclonal antibody was monitored through 52 h of production . Samples were automatically drawn from a bioreactor into the injection valve of an HPLC system without prior sample preparation . The hybridoma cell line was nonadherent, so whole cells were injected directly onto the perfusable protein A affinity column . There was only a modest column back pressure (ca . 1700 psi at a linear flow rate of 1.5 cm/s) after over 75 injections over the 52-h experiment . These experiments demonstrate the utility of high-speed chromatography for rapid process monitoring.

J Immunol, 1993 Dec 1, 151(11), 5948 - 54
Reversion of the SCID phenotype by human T cell grafts . Development of cross-species immunocompetence; Donjon CM et al.; Due to defective recombinase function, mice with severe combined immunodeficiency (SCID) lack functional lymphocytes and can accept human lymphoid xenografts . Xenografted animals (SCIDhum) are thought to provide a neutral environment for in vivo studies of normal, malignant or HIV-infected human cells . SCIDhum often develop endogenous, EBV+ lymphomas in the graft and in the our study two-thirds of 142 SCIDhum mice did so . Surprisingly, one-third of animals developed reversion of the SCID phenotype rapidly after human T cell engraftment . 90% of tumors occurred in nonrevertant and only 10% in revertant mice . These revertant animals showed immunologic tolerance for normal human B lymphocytes, maintained stable levels of mouse and human IgM and IgG . In addition, they generated competent mouse T cells able to kill transformed (EBV+) but not fresh B cells from the same donor nor unrelated human B cell lines . The tolerance for human lymphoid cells and the cross-species antitumor competence of host T lymphocytes imply unexpected recognition and selection events . Rather than a neutral "bioreactor," these observations mark the SCID host as potentially active participant in a composite immune system generated by xenografting.

Int J Artif Organs, 1993 Dec, 16(12), 843 - 6
Hepatocyte aggregate culture technique for bioreactors in hybrid liver support systems; Gerlach JC et al.; Utilizing a modified culture technique for hepatocytes, a high performance suspension culture is possible in which hepatocytes spontaneously form cell aggregates . The aggregates of 20-100 cells have been histologically confirmed to hold a three-dimensional structure, they show a long-term external metabolism and a survival time comparable with standard adhesion cultures . This technique has several advantages in the construction of large scale bioreactors for hybrid liver support systems.

Braz J Med Biol Res, 1993 Dec, 26(12), 1305 - 17
Preparation of human rabies vaccine in VERO cell culture using a microcarrier system; Mendonca RZ et al.; 1 . The rabies virus (Pasteur PV strain) was propagated in VERO cells attached to microcarriers in a 3.7-1 bioreactor . Virus titers of about 10(6) LD50/ml were obtained regularly . 2 . Ultrafiltration was efficient for concentrating the virus suspensions, and the sucrose gradient reduced the residual VERO cell DNA to acceptable levels (less than 50 pg/dose) . The remaining cell DNA content was evaluated by dot-blot hybridization with a probe prepared with VERO cell DNA . 3 . The final virus preparations were inactivated by B-propiolactone treatment, showed a potency higher than 2.5 IU/dose and protected mice experimentally infected intracerebrally with rabies virus (CVS-13.2) . 4 . This methodology for the production of a rabies vaccine for human use should be of interest to countries where high technology facilities are not available.

Analyst, 1993 Nov, 118(11), 1361 - 5
Simultaneous determination of ammonia nitrogen and L-glutamine in bioreactor media using flow injection; Palsson BO et al.; A novel split stream flow injection (FI) system suitable for the simultaneous determination of L-glutamine and ammonia nitrogen (ammonia-N) in cell culture media is described . Potentiometric detection of ammonia-N in one portion of the manifold is achieved using a commercial ammonia gas-sensing electrode fitted with a wall-jet cap . L-Glutamine is quantified in the other part of the split sample by potentiometric detection of ammonium ions (by an ammonium-selective polymer membrane electrode), liberated from the hydrolysis of glutamine after the sample flows through a glass bead reactor containing immobilized glutaminase . Endogenous ammonia-N and potassium ions that would normally interfere with the glutamine measurement are removed upstream using a unique tubular cation-exchange unit . Using 50 microliters sample volumes and mixed solutions of ammonium chloride and L-glutamine in Iscove's Modified Dulbecco's Medium to calibrate the FI measuring system, values for ammonia-N and L-glutamine determined for 22 media samples obtained from a bioreactor growing retroviral producer cells correlate well with those measured with commercial, manual enzymic-spectrophotometric assay kits.

Am J Surg, 1993 Nov, 166(5), 512 - 21
Evolution of the bioartificial liver: the need for randomized clinical trials; Nyberg SL et al.; The pursuit of a bioartificial liver is well documented in the literature . Early techniques of artificial liver support that have undergone clinical testing included simple exchange transfusions, extracorporeal xenogeneic or allogeneic liver perfusion, cross-circulation, hemodialysis, charcoal hemoperfusion, and plasmapheresis with plasma exchange . These techniques failed because they were unable to adequately support those hepatic functions essential for survival and because they lacked a back-up therapy, such as liver transplantation, for irreversible forms of liver disease . The concept evolved that hepatic functions essential for survival would be best performed by hepatocytes in an apparatus that allowed sustained or repetitive application . The best results have been achieved with bioartificial liver technologies that employ hepatocytes as implantable systems or extracorporeal devices . Implantable bioartificial liver systems include hepatocytes that have been on coated microcarrier beads, within microencapsulated gel droplets, within biodegradable polymeric substrates, or as spheroid hepatocyte aggregates . Extracorporeal systems include hepatocytes in suspension, on flat plates, and in hollow fiber bioreactors . Several extracorporeal systems have undergone extensive animal testing and are entering the early stages of human clinical trials . Randomized trials are needed to establish the value of bioartificial liver support in the treatment of patients with acute hepatic failure or as a bridge to liver transplantation.

Cell Transplant, 1993 Nov-Dec, 2(6), 453 - 60
Correction of bilirubin conjugation in the Gunn rat using hepatocytes immobilized in alginate gel beads as an extracorporeal bioartificial liver; Fremond B et al.; A new extracorporeal bioartificial liver using alginate-entrapped hepatocytes was developed and evaluated for its ability to correct the lack of bilirubin conjugation in the Gunn rat . Hepatocytes were harvested from Sprague-Dawley rats by the two-step collagenase perfusion method and then immobilized in Ca(++)-alginate beads . The ability of immobilized hepatocytes to conjugate bilirubin was investigated in vitro by comparison with hepatocyte monolayer cultures . The bioartificial liver consisted of a cylindric bioreactor containing either alginate beads with hepatocytes (test group) or alginate beads alone (control group) . Gunn rats were connected to this bioreactor via an extracorporeal circulation and bile fractions were collected at hourly intervals . Both bilirubin monoconjugates and bilirubin diconjugates were measured in the bile by high pressure liquid chromatography . Hepatocyte viability in alginate beads was determined prior to and at the end of each experiment and found to be unchanged (75%) . In the test group, the concentration of bilirubin conjugates increase rapidly, attaining median values of 72.26 microM and 92.59 microM for mono and diconjugated bilirubin respectively, during a 3 h period of extracorporeal circulation . In the control group, the levels of either conjugate did not exceed 0.87 microM throughout the experiments . Statistical analysis showed a significant difference between the two groups (p < 0.0023) . These results suggest that the bioartificial liver used in this study represents an effective method for the temporary correction of the Gunn rat's genetic defect . Such a system might be of therapeutic interest in acute liver failure.

Appl Microbiol Biotechnol, 1993 Nov, 40(2-3), 246 - 50
Immobilization of the slime mould Dictyostelium discoideum for the continuous production of recombinant human antithrombin III; Tiltscher H et al.; Recombinant vegetative Dictyostelium discoideum cells were immobilized inside a porous matrix by an inorganic membrane that was permeable to nutrients but not to cells, in order to produce recombinant human antithrombin III . Cells so entrapped could reach up to 15 times higher biomass densities compared with organisms growing freely in suspension . The high cell concentration maintained in the immobilized cell bioreactor caused an increase in specific and volumetric productivity . In continuous operation a maximum volumetric antithrombin productivity of 56 ng h-1 ml-1 catalyst bulk volume was attained at a dilution rate of 0.016 h-1 . In addition, the good retention of metabolic activity for several weeks as well as the convenient form of storage and regeneration of the catalytic system were shown.

Biotechnol Prog, 1993 Nov-Dec, 9(6), 675 - 8
Production of human alkaline phosphatase, a secreted, glycosylated protein, from a baculovirus expression system and the attachment-dependent cell line Trichoplusia ni BTI-Tn 5B1-4 using a split-flow, air-lift bioreactor; Chung IS et al.; A split-flow, air-lift bioreactor for the cultivation of insect cells to produce recombinant protein is described . It can be used advantageously with attached cell systems . This bioreactor incorporates two sections: a rise and a downcomer . Trichoplusia ni BTI-Tn 5B1-4 cells are grown on a support material of glass beads or microcarriers placed in the downcomer . This cell line is more productive than other commonly used insect cell lines, but it has the disadvantage of being difficult to use at large volumes since it is not easily adaptable to suspension culture . Adequate oxygen demand is supplied by sparging without direct exposure of cells to air bubbles . Nutrients are supplied convectively to the attached cells on support material as the fluid flows through the downcomer . The split-flow, air-lift bioreactor appears to be suitable for insect cell culture and is potentially scalable . It can provide a high surface-to-volume ratio and can be operated in batch or continuous mode . A lab-scale prototype bioreactor has been constructed and tested for the production of a secreted, glycosylated recombinant protein (human alkaline phosphatase) or seAP using an Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) vector . With a ratio of riser cross-sectional area to downcomer cross-sectional area of 1, an aspect ratio of 4.4, an air-flow rate of 54 mL/min in the riser, and a bed of 2400 3-min nonporous glass beads, 10.7 micrograms/mL of seAP was produced using an MOI (multiplicity of infection or the ratio of plaque-forming units to cells) of 10.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotechnol Prog, 1993 Nov-Dec, 9(6), 666 - 70
In situ fluorescence cell mass measurements of Saccharomyces cerevisiae using cellular tryptophan; Horvath JJ et al.; This work describes a new spectroscopic optical fiber/rod technique for in situ real time measurement of cell mass and product concentrations in bioreactors using intrinsic fluorescence . The variable excitation/emission wavelength capability of this sensor allows for species-selective measurement during fermentations . Cell mass (tryptophan) and product concentrations (pyridoxine) have been measured during fermentations of Saccharomyces cerevisiae . The effects of varying substrate concentration and oxygen concentration on the observed cell mass signals are eliminated by direct measurement of cell mass, as opposed to indirect measurement schemes such as those using NADH fluorescence . The sensor is robust and able to undergo many cycles of in situ steam sterilization without degradation, and its fluorescence signal is linear with concentration for all species studied in this work . Tryptophan fluorescence from yeast is shown to be a better measure of cell mass than NADH fluorescence.

Biotechnol Prog, 1993 Nov-Dec, 9(6), 580 - 6
Development of scale-down techniques for investigation of recombinant Escherichia coli fermentations: acid metabolites in shake flasks and stirred bioreactors; Dahlgren ME et al.; We have developed shake-flask screening conditions that are predictive of specific expression of the chimeric toxin, TGF alpha-PE40, by recombinant Escherichia coli JM109 in stirred bioreactors . When a nutrient-rich stirred bioreactor medium was used in shake flasks, neither the extent of growth nor the specific level of recombinant protein expression duplicated the performance in stirred bioreactor fermentations . Incomplete oxidation of glucose and concomitant accumulation of organic acid metabolites, as well as oxygen limitation and lack of pH control, were examined as contributors to the poorer performance in the flask . The medium buffering capacity, initial glucose level, and flask aeration were evaluated to establish the limits of "scale-down" conditions for expression both in a complex nutrient medium (M101) similar to that used in stirred bioreactors and in a defined (FM) medium . Acid metabolites and ethanol were measured as indicators of carbon flow from glucose as well as indirect indicators of oxygen limitation . For the complex M101 medium, optimal shake-flask performance in 250-mL, nonbaffled flasks at 37 degrees C occurred with 0.3 x medium strength, supplementation with 0.3 m HEPES buffer (pH 7.5), and 10 mL of medium per flask . Cultures grown under these conditions produced a maximum density of 3.6 g of dry cell weight/L (as estimated by absorbance measurements at 600 nm) and maintained a pH near neutrality . Additionally, metabolite markers of anaerobic or microaerobic conditions, such as ethanol, lactate, and pyruvate, were not detected, and specific expression of TGF alpha-PE40 was comparable to stirred bioreactors induced for expression at various biomass levels.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biotechnol, 1993 Nov, 31(2), 205 - 17
Fed-batch culture of insect cells: a method to increase the yield of recombinant human nerve growth factor (rhNGF) in the baculovirus expression system; Nguyen B et al.; A fed-batch method was developed which increased the density of insect cells (Spodoptera frugiperda, Sf-9 cells) in suspension culture and the feeding of nutrients improved the yield of a recombinant protein produced by a baculovirus expression system . Analysis of spent medium samples indicated that depletions of glucose and glutamine correlated with the retardation of cell growth . Feeding of a mixture of nutrients consisting of glucose, glutamine, yeastolate and lipids solution restored the growth rate . In fed-batch culture, cell density was increased from 3 x 10(6) cells per ml to 1.2 x 10(7) cells per ml and the increased cell density enhanced the yield of the desired recombinant product, in this case, human nerve growth factor (rhNGF) . The optimal conditions for the production of rhNGF were also defined by selecting the appropriate viral multiplicity of infection (MOI) . At a cell density of 5 x 10(6) ml-1, a MOI of 0.05 (plaque forming units per cell) gave the highest yield of rhNGF in culture fluid 3 d post-infection . The yield of rhNGF was 20 mg l-1 . The fed-batch method was scaled up to 12 l stirred bioreactor.

J Biotechnol, 1993 Nov, 31(2), 147 - 60
Search for cell proliferation markers suitable for cell count in continuous immobilized cell bioreactors; Capiaumont J et al.; The control of hybridoma cell cultures in bioreactors requires the use of convenient indicators to monitor the proliferation of the biomass . In order to select appropriate indications, we followed the variations of several compounds including tumoral markers, polyamines, sialic acids, purine and pyrimidine bases, enzymes and metabolites such as glucose, lactate and amino acids, and the variations of cell density during batch culture . Significant correlations were found between the number of viable cells and alkaline phosphatase, beta-glucuronidase, glucose and lactate measured in the culture medium of hybridoma strains . The correlation calculated from alkaline phosphatase and beta-glucuronidase concentrations in culture medium underestimated cell number . The correlation established with glucose and lactate gave the best indication of cell proliferation in continuous culture with an immobilized cell bioreactor . Finally, the exact quantification of the biomass in these culture conditions can be obtained using the mean of glucose and lactate correlations.

Enzyme Microb Technol, 1993 Nov, 15(11), 928 - 35
Continuous enzymatic production of oligopeptides: synthesis of an enkephalin pentapeptide in a multistage bioreactor; Richards AO et al.; A feasibility study of the continuous enzymatic production of short oligopeptides was undertaken using the synthesis of {Leu5}-enkephalin pentapeptides as a model system . A three-stage bioreactor was designed to perform the independent syntheses of the constituent tri- and dipeptide fragments and their subsequent condensation . Both the N-terminal (N-X-L-Tyr.Gly.GlyOEt) and C-terminal (L-Phe.LeuNH2) peptides were prepared in 75-85% yield in column reactors packed with Celite-immobilized alpha-chymotrypsin . The enkephalin pentapeptide was obtained in up to 30% yield in the third bioreactor module containing Celite-immobilized proteinase K . The bioreactor was run continuously for over 1,000 h, producing, under steady-state conditions, up to 0.7 g day-1 of the pentapeptide.

Am Biotechnol Lab . 1993 Nov;11(12):26.
Application of a hollow fiber membrane cell culture system in medicine; Marx U et al.; The Tecnomouse system is useful for cultivating transformed cell lines producing MAbs or recombinant proteins, but human tumor cells can also be propagated for autologous immunization protocols or research properties . Lymphokine-activated killer cell production and stem cell proliferation seem to be possible . Moreover, primary human cells of lymphoid organs can be successfully kept viable over long periods of time in a three-dimensional, tissue-like culture . Therefore, the bioreactor is a tool for the in vitro modulation of a variety of human organs.

Biotechnology (N Y), 1993 Nov, 11(11), 1263 - 70
Transgenic livestock as bioreactors: stable expression of human alpha-1-antitrypsin by a flock of sheep; Carver AS et al.; We have previously described the generation of transgenic sheep expressing human alpha 1-antitrypsin (h alpha 1AT) in their milk . Here, we report the fidelity of transgene transmission and expression by these animals and their progeny . Transgene transmission has been demonstrated in four of six ovine lines studied . Three of these four lines have exhibited stable transmission of the transgene, whereas the fourth has produced some offspring with reduced copy numbers . Sequential lactations of founder animals has yielded very similar levels of h alpha 1AT protein in milk . Moreover, in one line, derived from a founder male, a flock of seven G1 ewes have yielded comparable levels of h alpha 1AT protein in first and second lactation milk . Two G2 ewes of this line have also produced levels of human protein equivalent to their mother . Although the inheritance of the same transgene in mice was reminiscent of the situation in sheep, stable expression was observed in only one or four lines studied . The importance of these observations to the use of transgenic livestock as bioreactors for the production of human proteins is discussed.

Endocrinology, 1993 Oct, 133(4), 1490 - 503
Purification and characterization of recombinant human thyrotropin (TSH) isoforms produced by Chinese hamster ovary cells: the role of sialylation and sulfation in TSH bioactivity; Szkudlinski MW et al.; The biological significance of glycosylation variants of pituitary glycoprotein hormones remains controversial because of the indirect methods usually employed to determine carbohydrate composition or structure as well as the use of unreliable biological/immunological ratio to determine bioactivity . We have previously characterized recombinant human TSH (rhTSH) secreted by Chinese hamster ovary cells attached to microcarrier beads in a large scale bioreactor after stable transfection of hCG alpha and hTSH beta minigenes . In the present study rhTSH has been used as a model to determine structure-function relationships of different isoforms of glycoprotein hormones . We have now produced greater than 200 mg rhTSH using a hollow fiber bioreactor . The highly purified rhTSH produced in the hollow fiber bioreactor (rhTSH-N) as well as rhTSH commercially produced in a large scale bioreactor (rhTSH-G) were quantitated by immunoassays, receptor binding assay, and amino acid analysis and further characterized by a variety of physico-biochemical methods, including chromatofocusing and carbohydrate analysis . rhTSH-G, rhTSH-N, as well as pituitary human TSH (phTSH) have been separated by chromatofocusing on a Mono P column into several isoforms with different pI values . Compositional analysis of the fractions showed higher sialic acid content in the more acidic rhTSH-G fractions . phTSH acidic isoforms showed higher total sulfate and sialic acid contents than the more basic fractions . The bioactivities of various TSH isoforms based on rigorous quantitation of mass by amino acid analysis determined in three different FRTL-5 cell bioassays showed that the more basic and less sialylated fractions of rhTSH-G were more active than the more acidic fractions . In contrast to the in vitro data, highly sialylated and acidic rhTSH-G isoforms showed longer plasma half-lives and higher in vivo bioactivity than the basic forms . These results indicate that secreted rhTSH, similar to intrapituitary phTSH, exists as a mixture of charge isoforms that are related at least in part to the degree of sialylation . The degree of sialylation, highly dependent on the bioreactor production conditions, appears to be the major factor affecting the charge heterogeneity, MCR, and bioactivity of rhTSH.

J Pharm Biomed Anal, 1993 Oct, 11(10), 921 - 6
On-line monitoring of urea in effluent liquid during haemodialysis; Orellana A et al.; An analytical system specially built for on-line urea monitoring is reported . Measurements are carried out in the effluent of a haemodialysis machine . The measuring system employs the dialyser inflow stream as a carrier solution channel in a continuous fashion . The analyser periodically samples the outflow stream of the dialyser by means of an automatic injection valve . The analyser features a bioreactor consisting of immobilized urease and a gas-diffusion module . It is through this module that the urea is converted to ammonia gas which is transferred to another carrier channel, this transports the ammonium ion to a tubular, all-solid-state, ion-sensitive electrode . A timer controls the transport, injection, the measuring and the recording subsystems . The analyser has been used during actual haemodialysis sessions . Urea clearances were also measured in batch, using conventional spectrophotometric clinical equipment . The correlation between both methodologies was sufficient to confirm the usefulness of the developed on-line analyser to monitor the optimal length of haemodialysis sessions.

Arzneimittelforschung, 1993 Oct, 43(10), 1134 - 9
Mammalian cell cultures . Part I: Characterization, morphology and metabolism; Werner RG et al.; Primary cell cultures are obtained by trypsinization from tissue cultures usually as a monolayer culture . The absence of fetal calf serum will support suspended growth behaviour of spontaneously transformed cells . After several passages the cell line becomes more stable and gives rise to a continuous cell line . Such continuously growing cell lines are a prerequisite for production of recombinant DNA derived proteins . Mammalian cells are 10-100 times larger in diameter than microorganisms . They have no cell wall and express therefore a higher sensitivity to hydrodynamic sheer forces . One of the most stringent problems in mammalian cell culture are "silent" contaminants with mycoplasma which might change cell growth . Mammalian cell cultures show a complex metabolism where regulation of metabolites and catabolites are not fully understood . Glucose is the main carbohydrate source . Also three groups of intercorrelated amino acids are known . Lactate as the primary metabolite of glucose and ammonia as a metabolite of glutamine are expected to be cytotoxic for mammalian cells . Although in some experiments even the addition of ammonia has no significant effect on the viability of hybridoma cells . Adherent cells can be cultivated attached to surfaces such as microcarrier or wire springs . Suspended cells are grown in stirred bioreactors with a comparable technology to fermentation of microorganism . Parameters such as pH, temperature, stirring tip speed and osmolality have to be well controlled in order to obtain high cell viability and cell density.

Biotechniques, 1993 Oct, 15(4), 674 - 83
Pilot-scale production of murine monoclonal antibodies in agitated, ceramic-matrix or hollow-fiber cell culture systems; Kurkela R et al.; The purpose of this research was to compare three bioreactor systems for the pilot-scale production of monoclonal antibodies (MAbs) . We needed to produce gram quantities of murine MAbs against human prostatic acid phosphatase for use in fragmentation, radiolabeling and in vivo radio-imaging studies . The stable hybridoma cell line secreting IgG1 antibodies was chosen for production . Of the available bioreactor systems, we chose to test an agitating 30-liter bioreactor in repeated batch mode, a ceramic-matrix bioreactor in both repeated batch and continuous perfusion modes and a hollow-fiber bioreactor in continuous perfusion mode . The highest cultured MAb yield, 151 +/- 126 mg/day (mean +/- SD, n = 22), was achieved in the 30-liter bioreactor in repeated batch mode with the MAb being harvested in a large volume of medium, giving a reactor productivity of 4.3 +/ 3.4 mg/liter/day (mean +/- SD, n = 22) . The most concentrated MAb was harvested from the continuously perfused hollow-fiber bioreactor, which had the highest reactor productivity, 307 +/ 142 mg/liter/day (mean +/- SD, n = 47) and an average rate of MAb production of 55.3 +/- 25.7 mg/day (mean +/- SD, n = 47) . Taking the use of serum into consideration, the cost of MAb production was lowest in the continuously fed and harvested ceramic-matrix and hollow-fiber cell culture systems . A compact blood glucose meter proved to be a novel and suitable device for the rapid monitoring of glucose concentrations in hybridoma cultures.

Trends Biotechnol, 1993 Oct, 11(10), 413 - 8
Biocatalysis in the gas phase; Lamare S et al.; The biocatalysis of substrates in the gas phase may offer advantages over many conventional solution-based reactions, both in analytical devices and in bioreactors designed to accommodate this new technology . To date, however, the range of substrates for which gas-phase biocatalysis has been shown to be suitable is limited . Further research is required to establish the parameters that affect the kinetics and productivity of such systems.

Bioconjug Chem, 1993 Sep-Oct, 4(5), 341 - 6
Temperature-responsive bioconjugates . 2 . Molecular design for temperature-modulated bioseparations; Takei YG et al.; We have synthesized carboxyl semitelechelic oligo(N-isopropylacrylamide) (OIPAAm) using radical telomerization with 3-mercaptopropionic acid . This telomerization is also effective for the synthesis of carboxyl semitelechelic co-oligomers of IPAAm with butyl methacrylate (BMA) as hydrophobic or N,N-dimethylacrylamide (DMAAm) as hydrophilic comonomers . All co-oligomers are highly water-soluble at lower temperatures and exhibit phase separation with increasing temperature . Pure OIPAAm exhibits a lower critical solution temperature (LCST) at 32 degrees C, and the LCST for co-oligomers can be controlled to increase over 32 degrees C with increasing DMAAm composition and to decrease below 32 degrees C with increasing BMA composition . OIPAAm was grafted to bovine serum albumin (BSA) and bovine plasma fibrinogen (BPF) by activated ester-amine coupling . These OIPAAm-biomolecule conjugates maintain their temperature responses, are soluble in cold water, and precipitate over a range of temperatures related to oligomer content . Conjugates could be selectively precipitated and independently separated from conjugate solution mixtures with increasing temperature . In this case, the number of OIPAAm molecules attached to a conjugate affects the aggregate sizes of precipitated conjugates in mixtures . Both conjugate mixture ratios and solution concentrations influence the contamination of oligo(IPAAm-co-DMAAm)-BSA conjugates in precipitated oligo(IPAAm-co-BMA)-BPF conjugates . Furthermore, precipitated conjugates separated using centrifugation and filtration redissolve in water and maintain their biofunctionality, indicating the potential of strategy in reversible bioreactors and protein separations.

J Biochem Biophys Methods, 1993 Sep, 27(2), 105 - 16
A new NMR airlift bioreactor used in 31P-NMR studies of itaconic acid producing Aspergillus terreus; Lyngstad M et al.; An airlift bioreactor for in-vivo NMR studies of cells is described . The 10-mm diameter airlift reactor was constructed for studies of mycelial/pellet forming organisms, grown in suspension . With this device 161 MHz 31P-NMR spectra of living Aspergillus terreus cells, producing itaconic acid, have been obtained . Signals were observed for intra- and extracellular orthophosphate, glycerol-3-phosphorylethanolamine (GPE), glycerol-3-phosphorylcholine (GPC), sugar phosphates and polyphosphate . The spectra also showed broad overlapping resonances in the shift range of NAD(H) and NADP(H) . Polyphosphate disappeared when the respiratory gas was exchanged for pure N2 . The intracellular pH was estimated at 6.2 . In spectra of cell extracts approx . 60 peaks were observed in the range of 20 to -22 ppm, and they confirmed the appearance of the metabolites observed in living cells.

Biomaterials, 1993 Sep, 14(11), 803 - 8
Bioreactor applications of glucose oxidase covalently bonded on pHEMA membranes; Arica MY et al.; Glucose oxidase was immobilized onto poly(2-hydroxyethyl methacrylate) membranes by covalent bonding through epichlorohydrin . The highest immobilization efficiency was found to be 17.4% . The Km values were 5.9 and 8.8 mM for free and bound enzymes, respectively, and the Vmax values were 0.071 and 0.067 mM/min for free and bound enzymes . When the medium was saturated with oxygen Km was not altered significantly but Vmax was . The optimum pHs for the free and bound enzyme were determined to be 5 and 6, respectively, and the optimum temperature was 30 degrees C for both forms . The inactivation constant for the bound enzyme was found to be 1.7 x 10(-4) min-1.

J Biotechnol, 1993 Sep, 30(3), 299 - 313
Use of collagen hydrolysate as a complex nitrogen source for the synthesis of penicillin by Penicillium chrysogenum; Leonhartsberger S et al.; Optimal conditions for both biomass formation and penicillin synthesis by a strain of Penicillium chrysogenum were determined when using a collagen-derived nitrogen source . Preliminary investigations were carried out in shaken flask cultures employing a planned experimental program termed the Graeco-Latin square technique (Auden et al., 1967) . It was initially determined that up to 30% of a conventional complex nitrogen source such as cottonseed meal could be replaced by the collagen-derived nitrogen source without decreasing the productivity with respect to the penicillin yield . In the pilot scale experiments using a 30 l stirred tank type of bioreactor, higher penicillin yields were obtained when 70% of the conventional complex nitrogen source in the form of cottonseed meal was replaced by the collagen hydrolysate . Furthermore, the maximum rate of penicillin synthesis continued for over a longer period when using collagen hydrolysate as a complex nitrogen source . Penicillin synthesis rates were determined using a linear regression.

Enzyme Microb Technol, 1993 Sep, 15(9), 722 - 9
Two-liquid-phase bioreactors; Van Sonsbeek HM et al.; The application of two liquid phases that are poorly miscible is a fascinating research topic for biocatalytical conversions because of the promising results . Motives for application include an increase of productivity and achievement of continuous processing, but new limitations arise, e.g., interfacial effects such as biocatalyst accumulation and loss of activity, medium component accumulation, and slow coalescence . Centrifuges, membranes, and immobilization are tools that can overcome part of the problems, but more fundamental knowledge about interfaces and coalescence is still necessary for successful application . For scaleup and further development of processes based on the obtained results, a choice must be made for the configuration of the experimental setup of a bioreactor . Aspects like aeration, shear stress, batch or continuous processing, and immobilization can play an important role . This review article describes these aspects and the proposals that have been made in recent years concerning two-liquid-phase bioreactors . It shows some adaptations to existing bioreactors, such as loop reactors and stirred-tank reactors.

J Chromatogr, 1993 Aug 27, 646(1), 143 - 51
High-performance liquid chromatography of amino acids, peptides and proteins . CXXX . Modified porous zirconia as sorbents in affinity chromatography; Wirth HJ et al.; The utilisation of organosilanes to introduce active chemical groups onto zirconia surfaces, suitable for the subsequent immobilisation of proteins or other biomimetic ligands, is described . Two different types of porous zirconia-based particles with nominal pore diameters of 160 and 1000 A pore size were modified with two different affinity ligands . In the first case, methods to immobilise iminodiacetic acid-Cu(II) and its application in Cu(II) immobilised metal ion affinity chromatography (IMAC) were established . In the second series of experiments, concanavalin A was immobilised and the interaction of this lectin with the enzyme horseradish peroxidase examined . For both systems, adsorption isotherms were recorded as batch experiments . In each case, the experimental results could be fitted to langmuirean type adsorption isotherms, indicating that under the chosen conditions only one type of interaction is present, with nonspecific interactions with the support surface playing an insignificant role . These studies document the potential of surface modified zirconia particles for the immobilisation of chemical ligands or proteins for use in biospecific affinity chromatography and immobilised enzyme bioreactors.

Biotechnol Appl Biochem, 1993 Aug, 18 ( Pt 1), 1 - 8
Selective removal of beta-lactoglobulin directly from cow's milk and preparation of hypoallergenic formulas: a bioaffinity method; Chiancone E et al.; beta-Lactoglobulin, a major allergen of cow's milk, can pass into the breast milk of mothers consuming dairy products and thus sensitize a predisposed infant with a family history of atopic allergy . beta-Lactoglobulin coupled to Sepharose 4B can be used as a specific affinity matrix to remove beta-lactoglobulin from milk without the use of buffer systems . The tendency of beta-lactoglobulin to polymerize reversibly in milk promotes binding of the soluble protein to the coupled protein and a significant retardation in its elution with respect to the other milk proteins . The matrix can be regenerated with water and used repeatedly . These unique characteristics can be exploited in bioreactors designed to treat milk for the preparation of new hypoallergenic hyposensitizing milk formulas to be used by lactating mothers.

Int J Artif Organs, 1993 Aug, 16(8), 604 - 8
Side effects of hybrid liver support therapy: TNF-alpha liberation in pigs, associated with extracorporeal bioreactors; Gerlach J et al.; During acute liver failure, hybrid liver support therapy could serve as a bridge to liver transplantation . In this desired temporary use, immune competent cell responses, such as the production of cytokines, might be of limiting relevance . We have investigated the Tumor Necrosis Factor-alpha (TNF) liberation in two models using pigs, connected with an extracorporeal bioreactor with homologous hepatocytes: TNF liberation was measured in arterial plasma during a 4 day perfusion time in untreated animals, model (i), and during short term perfusion of hepatectomized pigs in model (ii) . Animals four days after catheter implantation in model (i) had TNF values of < 5 pg/ml . After connecting the system without hepatocytes, TNF rose to 9.7 +/- 2 within 120 min and rose further to 32.6 +/- 6 pg/ml within 4 hours after filling the system with the homologous hepatocytes . After 24 hours of continuous perfusion and during four days of perfusion, the TNF levels were lowered to baseline levels . In model (ii), TNF rose to 220 +/- 130 pg/ml within 180 min and decreased to 110 +/- 10 pg/ml within six hours, whereas controls without hepatocytes showed mean levels with a maximum of 120 +/- 20 pg/ml . In both models, there was no correlation between TNF levels and clinical abnormalities such as fever or shock symptoms . There is evidence for an activation of blood cells during experimental extracorporeal hybrid support . No typical side effects were, however, observed . Thus, TNF mediated extracorporeal cell activation does not appear to limit the application of homologous hybrid liver support therapy.

J Biotechnol, 1993 Aug, 30(2), 231 - 43
High level expression of alpha-human atrial natriuretic factor as a fusion polypeptide with phage fr coat protein in Escherichia coli; Berzins V et al.; A synthetic DNA sequence coding for the 28 amino acid residues of alpha-human atrial natriuretic factor (alpha-hANF) and the N-terminal linker tripeptide Ile-Asp-Lys was inserted in the 3'-terminal part of the RNA bacteriophage fr coat protein gene . The cloned hybrid gene was isolated and placed into an expression vector under the control of the inducible E . coli tryptophan promoter and phage fr coat protein translation initiation region (TIR) sequence . In an appropriate host strain the expressed fusion protein accounts for at least 10% of the total cellular protein . In order to achieve high-cell density in a bioreactor while maintaining efficiency of alpha-hANF expression, improved cultivation conditions were selected using modified Shielach-Bauer's culture media containing glucose, yeast extract and bacto tryptone at an initial concentration of 2 g l-1 of each, adding concentrate of medium throughout the microbial growth and maintaining the dissolved oxygen in a range of 25-30% . At 13-14 h cultivation, the cell density reached 40 g cell dry weight per liter and the yield of fusion protein exceeded 45 mg g-1 cell dry weight . Fusion protein from solubilized E . coli cells was purified to homogeneity by ion exchange chromatography on DEAE-, CM-cellulose, QAE Sephadex A25 columns and selective precipitation.

Blood, 1993 Jul 15, 82(2), 378 - 84
Large-scale expansion of human stem and progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures; Koller MR et al.; There is a growing consensus that clinical practice in the areas of bone marrow (BM) transplantation and gene therapy will rely on the ex vivo expansion of hematopoietic cells . We report here on the development of continuously perfused culture systems (bioreactor systems) that expand human stem and progenitor cells from BM mononuclear cell (MNC) populations obtained without cell enrichment . In three separate experiments, 10 bioreactors were each inoculated with 3 x 10(7) BM MNC from patients undergoing marrow harvest for autologous transplantation . At various times thereafter (between days 6 and 16), duplicate bioreactors were harvested and cells were analyzed . The bioreactors contained three cell populations that were analyzed separately: nonadherent cells; cells that were loosely adherent to the endogenously formed stromal layer; and an adherent cell layer that required trypsinization for removal . Total cell numbers increased continuously to give an overall 10-fold (range, 8- to 11-fold) expansion by day 14 . The adherent stromal layer significantly expanded to more than 2 x 10(7) cells, but remained less than 6% of the total cell population . Colony-forming unit-granulocyte-macrophage (CFU-GM) numbers expanded 21-fold (range, 12- to 34-fold) by day 14 and, because this expansion was greater than that for total cells, CFU-GM were enriched by as much as fourfold by day 14 . Burst-forming unit-erythroid (BFU-E) numbers peaked earlier than did CFU-GM numbers, with a 12-fold (range, 6- to 18-fold) expansion obtained on day 8 . In contrast to CFU-GM, which were predominantly nonadherent, BFU-E were more evenly distributed between the three cell populations . Stem cell activity was measured by the long-term culture-initiating cell (LTC-IC) limiting dilution assay . The number of LTC-IC per reactor consistently increased with time in all cultures, resulting in a 7.5-fold (range, 3.4- to 9.8-fold) expansion . In summary, more than 3 billion cells, containing 12 million CFU-GM, were reproducibly generated from the equivalent of a 10 to 15 ml BM aspirate . These data indicate that small numbers of BM MNC can be readily expanded ex vivo in continuous perfusion cultures, and that such ex vivo expansion may have direct applications in clinical and experimental BM transplantation.

Anal Chem, 1993 Jul 1, 65(13), 1658 - 61
Simultaneous kinetic-based determination of fructose and ascorbate with a rotating bioreactor and amperometric detection: application to the analysis of food samples; Matsumoto K et al.; A recently introduced biosensor comprising a rotating bioreactor and a stationary platinum ring amperometric detector (Anal . Chem . 1993, 65, 636-639) has been utilized for the simultaneous determination of fructose and ascorbate . The approach has been successfully applied to the simultaneous determination of these analytes in fresh food samples . Hexacyanoferrate(II) is the monitored species at +0.380 V vs a Ag/AgCl, 3 M NaCl reference . The determination takes advantage of a fast chemical oxidation of ascorbate by hexacyanoferrate(III) ions and the subsequent slower production of hexacyanoferrate(II) in the D-fructose 5-dehydrogenase-catalyzed reaction between D-fructose and hexacyanoferrate(III) as acceptor . D-Fructose 5-dehydrogenase (EC 1.1.99.11) was incorporated into the rotating disk reactor in immobilized form and on controlled-pore glass via the glutaraldehyde attachment and cross-linking with bovine serum albumin . Sample/reagent processing was accomplished by programmed continuous-flow/stopped-flow/continuous-flow operation.

Artif Organs, 1993 Jul, 17(7), 653 - 9
Isolation and culture of human hepatocytes from resected liver tissue as a bioreactor for a hybrid artificial liver; Takahashi M et al.; To use cultured human hepatocytes as a hybrid artificial liver, effective methods for isolating and culturing the hepatocytes from resected surgical specimens were investigated . Two different procedures for isolating hepatocytes, perfusion and agitation with a collagenase solution (Method 1) and perfusion with a mixed solution of collagenase and dispase (Method 2), were examined . The yield of isolated hepatocytes obtained by Method 2 (13.31 x 10(6) cells/g of liver) was significantly higher than that by Method 1 (0.94 x 10(6)) . The warm ischemia time (0-90 min) of the liver fragments obtained did not disturb the viability and yield of the isolated hepatocytes . The gluconeogenesis and urea synthesis of the cultured human hepatocytes were well preserved for 10 days . These results show that for prolonged human hepatocyte culture (10 days), isolation from resected human liver tissues by a combination of the proteolytic enzymes collagenase and dispase was effective and warm ischemia was tolerated for up to 90 min, which indicates the possibility of using cultured human hepatocytes as a hybrid artificial liver.

ASAIO J, 1993 Jul-Sep, 39(3), M242 - 6
Immunologic considerations in the use of cultured porcine hepatocytes as a hybrid artificial liver . Anti-porcine hepatocyte human serum; Takahashi M et al.; Cultured porcine hepatocytes with reasonable metabolic functions are a promising bioreactor for a hybrid artificial liver in the treatment of liver failure . A cytotoxic human serum (t-serum) against cultured porcine hepatocytes was accidentally discovered during preliminary experiments, however, which prompted the authors to study the mechanism of the cytotoxicity and the frequency of occurrence of the cytotoxic sera . Among 103 individual sera examined, seven (6.8%) sera showed cytotoxicity to cultured porcine hepatocytes . T-serum heated at 56 degrees C for 30 min completely lost its cytotoxic activity, but the inactivated cytotoxicity was restored by the addition of rabbit complement to the inactivated t-serum . IgM-depleted t-serum produced by 2-mercaptoethanol treatment abolished the cytotoxicity to porcine hepatocytes . The cytotoxic reactions were therefore thought to be mediated by IgM capable of complement activation . Although IgM binding to porcine hepatocytes was also seen in nontoxic sera, the IgM did not activate complement . This implies differences in hepatocyte surface antigens recognized by IgM from t- and nontoxic sera . Before clinical application of a hybrid artificial liver using cultured porcine hepatocytes, detection of cytotoxic human IgM against porcine hepatocytes is necessary, and the means of eliminating the cytotoxic IgM from the hybrid artificial liver system, or of inactivating complement in the system, will be desirable in cases positive for cytotoxic IgM.

Appl Biochem Biotechnol, 1993 Jul, 42(1), 53 - 66
An inexpensive optical probe for measuring the local specific interfacial area; Yang JD et al.; An in situ optical probe was developed to measure reliably the local specific interfacial area of the suspended phase (specifically air bubbles) in a bioreactor . The light transmission-based probe can be simply and inexpensively constructed from readily available components . The probe's performance was tested in a suspension of opaque monodisperse polystyrene spheres as well as in the presence of nonspherical, nonuniformly distributed bubbles . The probe signal is directly related to the local specific interfacial area by a calibration equation obtained with polystyrene beads, as opposed to the cumbersome direct photographic bubble measurements that the probe attempts to replace . Its utility was demonstrated by measuring the specific bubble interfacial area at two locations in a bioreactor at various agitation intensities.

Appl Microbiol Biotechnol, 1993 Jul, 39(4-5), 632 - 6
Stability and activity of a phenol oxidase from the ligninolytic fungus Pleurotus ostreatus; Palmieri G et al.; Three different phenol oxidases produced by the basidiomycete fungus Pleurotus ostreatus have been isolated and their main structural, enzymatic and physico-chemical properties characterized . Studies have focused on the most abundantly secreted of these proteins, a copper-enzyme specific towards ortho-diphenol substrates . This protein was purified to homogeneity and part of its primary structure determined by direct protein sequencing . The influence of pH, temperature and presence of water-soluble or water-insoluble organic solvents on the activity and stability of the enzyme were also investigated . These data can be used for applying bioreactors to problems of environmental concern such as waste-water treatment.

Biotechnol Prog, 1993 Jul-Aug, 9(4), 355 - 61
Effects of oxygen/glucose/glutamine feeding on insect cell baculovirus protein expression: a study on epoxide hydrolase production; Wang MY et al.; The recombinant protein yields for batch cultures of the insect cell baculovirus expression system have been significantly enhanced by oxygen, glucose, and glutamine feeding . The improvement in both volumetric and specific yields was based on influencing the metabolism of infected cells . Oxygen was absolutely required for viral replication and high protein expression in infected cells . Increases of 200% in volumetric yield and 100% in specific yield of recombinant epoxide hydrolase were achieved by controlling the dissolved oxygen (DO) level to near 35% saturation . An additional 100% increase was achieved by glucose and glutamine feeding . Results indicated that the intracellular metabolite pool was not adequate for recombinant protein overproduction . Finally, the specific protein yield, based on initial infection cell density, in high cell density spinner flasks and bioreactors of spent media with glucose and glutamine feeding was equivalent to that freshly diluted cultures.

Mol Immunol, 1993 Jun, 30(8), 741 - 8
Control of IgG/Fc glycosylation: a comparison of oligosaccharides from chimeric human/mouse and mouse subclass immunoglobulin Gs; Lund J et al.; Oligosaccharide profiles were obtained for chimeric mouse-human antibodies corresponding to each of the human IgG subclasses 1-4, and mouse IgG2b antibodies each expressed in the mouse J558L cell line . These antibodies have specificity for the NIP hapten and form a matched set of IgGs . An IgG4 chimeric antibody (B72.3) produced in the chinese hamster ovary (CHO-K1) cell line was also analysed for carbohydrate . Additionally aglycosylated mutants of this IgG4 (B72.3) and anti-NIP mouse IgG2b were analysed . The total lack of carbohydrate found in the aglycosylated site-directed mutants human chimeric IgG4 B72.3 (Asn 297-->Gln) and mouse IgG2b (Asn 297-->Ala) demonstrates that there are no N-glycosylation sites other than Asn 297 . Therefore glycosylation profiles for all the IgGs analysed reflect carbohydrate attached to this site . Factors such as cell type (A), template direction by the IgG heavy chains (B) and culture conditions (C) are shown to influence IgG glycosylation profiles . (A) The anti-NIP IgG antibodies expressed by the J558L cell line may have one or two Gal (alpha 1-->3) Gal residues per oligosaccharide unit, indicative of the presence of (alpha 1-->3) galactosyl transferase in the J558L mouse cell line . (B) The galactosylation profiles obtained for the IgG heavy chains, in particular the preference for galactosylation of the Man (alpha 1-->6) arm rather than the Man (alpha 1-->3) arm, contrary to the beta-galactosyltransferase specificity, suggest that the polypeptide chain may act as a template to influence the extent of galactosylation and hence the proportions of each oligosaccharide incorporated . The IgG2 antibody does not display this galactosylation preference . (C) The extent of galactosylation appears to be influenced by the growth conditions, with the highest levels of galactosylation being found for IgG produced by cells grown in still cultures, rather than cells grown as ascites or in hollow fibre bioreactors . It is concluded that though the profile of glycosylation is controlled predominantly by the glycosylation activity of the cell in which the IgG is expressed, differences between the IgG heavy chain templates of the various subclasses and culture conditions can also influence glycosylation.

Appl Microbiol Biotechnol, 1993 Jun, 39(3), 329 - 34
Studies on the production of human parathyroid hormone by recombinant Escherichia coli; Harder MP et al.; Expression of human parathyroid hormone, hPTH(-1-84), by Escherichia coli N4830:pEX-PPTH was studied in controlled bioreactors . The hPTH is expressed as a fusion protein under control of the bacteriophage lambda PR promoter . In batch runs, low biomass concentrations but high specific hPTH productivities were obtained with complex TY (bactotryptone and yeast extract) medium whereas high biomass concentration and low specific productivities were found when fructose was used instead of bactotryptone (YF medium) . The preinduction temperature was always 30 degrees C; the temperature shift to induce production of fusion protein was varied from 36 to 42 degrees C . Formation of hPTH passed a pronounced maximum as a function of induction temperature when using YF medium . However, the optimum temperature shift was 38 degrees C for both media used . For this temperature increase both media yielded about the same volumetric hPTH productivity (approx . 30 mg hPTH/1 per hour) . By applying a fed-batch strategy for the YF medium, the productivity of the recombinant protein could be further increased more than fourfold . Compared to shake-flask experiments, the hPTH yield could be increased by a factor larger than 20.

Enzyme Microb Technol, 1993 Jun, 15(6), 525 - 9
Electrochemical bioreactor with immobilized glucose-6-phosphate dehydrogenase on the rotating graphite disc electrode modified with phenazine methosulfate; Miyawaki O et al.; Physical adsorption, covalent binding through the carbodiimide reaction between the surface carboxyl group and the amino group of the protein, and the crosslinking method with bovine serum albumin by glutaraldehyde were applied for the immobilization of glucose-6-phosphate dehydrogenase (G6PDH) on a graphite electrode . Among those, the crosslinking method was employed for its highest apparent enzyme activity per unit surface area . Phenazine methosulfate (PMS), as a mediator for the electrochemical oxidation of NADH, was also immobilized on the graphite surface through adsorption . The conjugation reaction of G6PDH and the electrochemical oxidation of NADH were confirmed by cyclic voltammetry and the constant potential electrochemical reaction . An electrochemical bioreactor system was established by using a rotating disc graphite electrode with G6PDH immobilized . The coenzyme, NAD, was effectively recycled between the electrochemical and the enzymatic reactions.

Enzyme Microb Technol, 1993 Jun, 15(6), 476 - 82
Thermal cycling effects on the bioreactor performances of immobilized beta-galactosidase in temperature-sensitive hydrogel beads; Park TG et al.; The enzyme beta-galactosidase was immobilized in thermally reversible hydrogel beads that exhibit a reversible expansion and collapse of the gel volume in response to temperature . The kinetic performances of immobilized enzyme bead reactors were studied during thermal cycling operation . A periodic cycling of temperature for the packed-bed reactor induced a cyclic swelling and deswelling of the hydrogel beads . A temperature cycling operation around the phase transition temperature of the gel matrix enhanced the overall enzymatic conversion of the substrate, compared to its upper and lower isothermal operations . The effects on the overall conversion of various thermal cycling operational conditions, such as cycling range and heating and cooling rates, were investigated.

J Immunol Methods, 1993 May 26, 161(2), 145 - 50
Production of bi-specific monoclonal antibodies in a hollow-fibre bioreactor; Gorter A et al.; In the present study we have measured the feasibility of producing high amounts of bi-specific monoclonal antibodies (MAb) for therapeutic purposes using a hollow-fibre bioreactor . We have studied the isotype composition and functional capacity of an OKT3/OV-TL 3 quadroma (IgG2a/IgG1) and we have compared the production of bi-isotypic MAb in this system with tissue culture flasks or mouse ascites . Both culture supernatant and purified bi-isotypic MAb were able to enhance the cytolytic activity of a CD8+ T cell clone against ovarian tumour cell lines, demonstrating the presence of functional bi-isotypic MAb (bi-specific MAb) . The total amount of immunoglobulin produced after 38 days was 375 mg . After purification by protein A affinity chromatography, 79 mg of bi-isotypic MAb (IgG1/IgG2a) was obtained . This amount of bi-isotypic MAb was similar to the amount obtained from ascitic fluid produced by 200 mice or 38 l of tissue culture flask supernatant.

Biochemistry, 1993 May 4, 32(17), 4665 - 70
Dimethyl methylphosphonate (DMMP): a 31P nuclear magnetic resonance spectroscopic probe of intracellular volume in mammalian cell cultures; Barry JA et al.; Dimethyl methylphosphonate (DMMP), when added to a suspension of erythrocytes, has been reported to have a lower frequency chemical shift inside of cells than outside . This work further investigates the same phenomenon in hollow-fiber bioreactor cultures of six mammalian cell lines and describes the application of DMMP as a measure of intra- versus extracellular volumes in mammalian cell cultures . No toxic effects of the DMMP were observed at the concentrations used here . The dependence of the shift of intracellular DMMP on intracellular protein content was shown to be similar for cultured mammalian and red blood cells . Also consistent with shifts in erythrocytes, an increase in the intracellular protein concentration due to a reduction in cultured cell volume increased the magnitude of the shift to lower frequency . Longitudinal relaxation (T1) values for intra- and extracellular DMMP were measured so that partially saturated DMMP peaks in 31P NMR spectra of mammalian cell cultures can be corrected to give the relative volumes of the intra- and extracellular compartments; this information provides a relative measure of culture growth . Intracellular volume measured by this method can also be used to quantify intracellular metabolites such as ATP during the growth of the culture . To explore the mechanism behind the intracellular shift, we have also addressed the three possible contributions to the chemical shift of DMMP: hydrogen-bonding interactions, magnetic susceptibility, and ionic strength . Data is presented which eliminates the latter two mechanisms and strongly supports the hypothesis that the observed intracellular shift is due to a reduction in hydrogen bonding between water and DMMP in the cytoplasm.

Res Microbiol, 1993 May, 144(4), 327 - 32
Measurement of the net production of acidity by a sulphate-reducing bacterium: experimental checking of theoretical models of microbially influenced corrosion; Daumas S et al.; The net production and consumption of acidity by Desulfovibrio fructosovorans, growing with lactate as the carbon and energy source, were measured in an unbuffered medium in a pH-controlled bioreactor . At alkaline pH (7.2 and 8.5), net acidity production was measured . At pH 6.0, acidity consumption was obtained, although bacterial growth was not observed . These observations are in good agreement with theoretical predictions emphasizing the key role of H+ ions in the relationship between the metabolism of sulphate-reducing bacteria and microbially influenced corrosion.

J Biotechnol, 1993 May, 29(1-2), 57 - 74
Automatic bioprocess control . 4 . A prototype batch of Saccharomyces cerevisiae; Locher G et al.; The recent investigations in our high performance bioreactors have shown that living cells can be extremely sensitive to physical-chemical environmental conditions and their changes . Consequently, the relationship bioreactor-living cell must thoroughly be investigated in order to discuss both: whether bioreactor characteristics are limiting/dominating during cultivation and to what extent controlled changes of the cellular environment can lead the cells to a desired physiological state . For these investigations, a generally accepted biological test organism would be helpful, of which the requirements and reactions under certain conditions are well known . Saccharomyces cerevisiae is a well known, very robust but nevertheless sensitive organism, eligible for this purpose . In this article a typical batch cultivation on glucose is presented, collected from approx . 300 experiments . Regarding metabolite production and consumption, seven different phases are distinguished on the basis of approx . 20 sensor signals and their metabolic background is discussed . Prerequisite, however, was an exhaustive knowledge upon extracellular conditions, a task which could successfully be fulfilled with the highly automated equipment introduced in the preceding articles of this series.

J Biotechnol, 1993 May, 29(1-2), 145 - 56
Influence of ammonium ion and glucose on mAb production in suspension and fixed bed hybridoma cultures; Racher AJ et al.; Previous work indicated that mAb production by a mouse-mouse hybridoma, grown in a fixed bed of macroporous glass beads with a variable feed glutamine concentration, was correlated only to QvO2 . The work reported in this study further investigated the metabolic parameters modulating mAb production using metabolic data from a continuous stirred tank reactor (CSTR) to interpret the behaviour of cells grown in a fixed bed bioreactor (FBR) . For a FBR culture grown with a feed glutamine concentration of 3 mmol l-1, QvmAb was correlated to QvO2 and QvGln . However, if the feed glutamine concentration was switched between 1 and 3 mmol l-1, the above relationship did not hold, probably because QvO2 was at or near its maximum value . For both the FBR and CSTR, switches in the feed glutamine concentration suggested that maximum QvmAb values were associated with glucose concentrations above 12.8 mmol l-1 and an ammonium concentration of 2.0-2.5 mmol l-1.

Biotechnol Prog, 1993 May-Jun, 9(3), 309 - 16
Fractional factorial study of hybridoma behavior . 2 . Kinetics of nutrient uptake and waste production; Gaertner JG et al.; A fractional factorial experimental method was used to identify important variables and variable interactions which affect nutrient uptake and waste production . The variables studied include glucose, glutamine, base medium, lactate, and ammonium . Cellular glucose uptake was stimulated by glucose and glutamine and inhibited by lactate . Glucose, base medium, and glutamine all had large stimulative effects on lactate production . Glucose uptake and lactate production both exhibited Monod-type dependencies on the glucose concentration . Ammonium production was sensitive to the glucose and lactate concentrations at levels below about 8 and 20 mM, respectively . Mathematical descriptions of glucose uptake and of lactate and ammonium production were developed . These experimental results have significant implications for the optimization of hybridoma growth in bioreactors.

Biotechnol Prog, 1993 May-Jun, 9(3), 298 - 308
Fractional factorial study of hybridoma behavior . 1 . Kinetics of growth and antibody production; Gaertner JG et al.; A comprehensive approach was taken toward the quantitative study of hybridoma growth and antibody production . A fractional factorial experimental method was used to identify important variables and variable interactions affecting hybridoma behavior . The variables studied include temperature, pH, dissolved oxygen, glucose, glutamine, base medium, serum, lactate, and ammonium . The growth rate was strongly affected by the levels of dissolved oxygen, pH, temperature, and base medium . Interactions between temperature, pH, and dissolved oxygen were important . The optimal pH for growth depends upon the temperature and dissolved oxygen concentration . In general, growth was fastest at low dissolved oxygen levels . The growth rate was very sensitive to low concentrations of base medium, but was relatively insensitive to the serum concentration at levels above 2.5% . Antibody production was stimulated by high concentrations of base medium and serum and inhibited by ammonium and lactate . Antibody production increased linearly with serum concentration . In general, conditions that favored a high growth rate also favored a high specific rate of antibody production . The functional dependencies of antibody production on base medium and ammonium were similar to those for cell growth; however, antibody production and cell growth exhibited different dependencies on serum . Mathematical descriptions of cell growth and antibody production were developed . These experimental results have significant implications for the optimization of hybridoma growth in bioreactors.

Int J Artif Organs, 1993 Apr, 16(4), 218 - 28
Extracorporeal enzymatic removal of low density lipoproteins in rabbits: efficacy and safety; Shefer SD et al.; An extracorporeal circuit incorporating a plasma separator reactor (PSR) was designed to modify low density lipoproteins (LDL) . The PSR was tested in vivo with hypercholesterolemic New Zealand White rabbits . The bioreactor enzymatically converts LDL to a form that can be removed by the body at an enhanced rate . The physiological response of hypercholesterolemic New Zealand White rabbits to 90 minute extracorporeal treatments was monitored . The total plasma cholesterol concentration in the treated rabbits fell sharply (up to 40% decrease) during and following the treatment . Results of safety tests indicate no significant enzyme leaching from the device, no disruption or damage to erythrocytes, no increase in white blood cell count and no liver damage as indicated by five enzyme assays . All safety measurements suggest that the treatment is safe.

Trends Biotechnol, 1993 Apr, 11(4), 136 - 42
Sampling and sample handling--crucial steps in process monitoring and control; Mattiasson B et al.; Sampling and sample handling are important considerations in the design of monitoring and control systems for bioreactors . Many factors must be considered in developing strategies for obtaining representative samples, thus ensuring that the status of the bioprocess can be evaluated rapidly, in a reproducible and reliable manner.

Curr Opin Biotechnol, 1993 Apr, 4(2), 205 - 10
Cultivation of roots in bioreactors; Curtis WR; Over the past few years, the number of reports of roots cultured in reactors has increased dramatically . This has provided much-needed information on the physical and metabolic requirements of cultured roots, and has established techniques for the manipulation of root tissue . Most of this work, however, is conducted on a relatively small scale (less than 5 l), and many are demonstrations as opposed to systematic studies . The real challenge of scale-up will be encountered when moving to the 100-1000 l scale, while maintaining dry tissue densities in excess of 10-20 (g dry weight) l-1 . Achievement of this goal will require small-scale studies, focused on obtaining design and operational information, coupled with measurements of fundamental scale-up parameters in large-scale systems.

Biotechnology (N Y), 1993 Apr, 11(4), 512 - 5
Comparative biochemical characterization of a human IgM produced in both ascites and in vitro cell culture; Monica TJ et al.; We have conducted a comparative analysis of a monoclonal human IgM obtained from cells cultured in nude-mouse ascites and from the same cells cultured in a bioreactor . We studied the glycosylation of the IgMs using lectin blotting and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD), and we also developed reverse phase liquid chromatography (RPLC) peptide maps of the IgM samples . The HPAE-PAD data indicate that the samples differ in both the type and distribution of oligosaccharides present on the IgMs . In addition, the proteins differ in their solubility behavior and in their RPLC peptide maps . We conclude that the method of cell culture is capable of significantly altering the characteristics of the glycoprotein product.

J Cell Biochem, 1993 Mar, 51(3), 290 - 300
Prospects for use of microgravity-based bioreactors to study three-dimensional host-tumor interactions in human neoplasia; Jessup JM et al.; Microgravity offers unique advantages for the cultivation of mammalian tissues because the lack of gravity-induced sedimentation supports three-dimensional growth in batch culture in aqueous medium . Bioreactors that simulate microgravity but operate in unit gravity provide conditions that permit human epithelial cells to grow to densities approaching 10(7) cells/ml on microcarriers in suspension, in masses up to 1 cm in diameter, and under conditions of low shear stress . While useful for many different applications in tissue culture, this culture system is especially useful for the analysis of the microenvironment in which host matrix and cells interact with infiltrating tumor cells . Growth in the microgravity-based bioreactor has supported morphological differentiation of human colon carcinoma cells when cultured with normal human stromal cells . Furthermore, these co-cultures produced factors that stimulated goblet cell production in normal colon cells in an in vivo bioassay . Early experiments also suggest that the microgravity environment will not alter the ability of epithelial cells to recognize and associate with each other and with constituents of basement membrane and extracellular matrix . These findings suggest that cells grown in bioreactors that simulate aspects of microgravity or under actual microgravity conditions will produce tissues and substances in sufficient quantity and at high enough concentration to promote characterization of molecules that control differentiation and neoplastic transformation.

J Cell Biochem, 1993 Mar, 51(3), 274 - 82
Studies of chondrogenesis in rotating systems; Duke PJ et al.; A great deal of energy has been exerted over the years researching methods for regenerating and repairing bone and cartilage . Several techniques, especially bone implants and grafts, show great promise for providing a remedy for many skeletal disorders and chondrodystrophies . The bioreactor (rotating-wall vessel, RWV) is a cell culture system that creates a nurturing environment conducive to cell aggregation . Chondrocyte cultures have been studied as implants for repair and replacement of damaged and missing bone and cartilage since 1965 {Chesterman and Smith, J Bone Joint Surg 50B:184-197, 1965} . The ability to use large, tissue-like cartilage aggregates grown in the RWV would be of great clinical significance in treating skeletal disorders . In addition, the RWV may provide a superior method for studying chondrogenesis and chondrogenic mutations . Because the RWV is also reported to simulate many of the conditions of microgravity it is a very useful ground-based tool for studying how cell systems will react to microgravity.

J Cell Biochem, 1993 Mar, 51(3), 265 - 73
Use of microgravity bioreactors for development of an in vitro rat salivary gland cell culture model; Lewis ML et al.; During development, salivary gland (SG) cells both secrete factors which modulate cellular behavior and express specific hormone receptors . Whether SG cell growth is modulated by an autocrine epidermal growth factor (EGF) receptor-mediated signal transduction pathway is not clearly understood . SG tissue is the synthesis site for functionally distinct products including growth factors, digestive enzymes, and homeostasis maintaining factors . Historically, SG cells have proven difficult to grow and may be only maintained as limited three-dimensional ductal-type structures in collagen gels or on reconstituted basement membrane gels . A novel approach to establishing primary rat SG cultures is use of microgravity bioreactors originally designed by NASA as low-shear culture systems for predicting cell growth and differentiation in the microgravity environment of space . These completely fluid-filled bioreactors, which are oriented horizontally and rotate, have proven advantageous for Earth-based culture of three-dimensional cell assemblies, tissue-like aggregates, and glandular structures . Use of microgravity bioreactors for establishing in vitro models to investigate steroid-mediated secretion of EGF by normal SG cells may also prove useful for the investigation of cancer and other salivary gland disorders . These microgravity bioreactors promise challenging opportunities for future applications in basic and applied cell research.

J Cell Biochem, 1993 Mar, 51(3), 257 - 64
Cultivation of cell-polymer cartilage implants in bioreactors; Freed LE et al.; Cartilage implants for potential use in reconstructive or orthopedic surgery can be created by growing isolated cartilage cells (chondrocytes) in vitro on synthetic, biodegradable polymer scaffolds . The scaffolds provide specific three-dimensional structures which support cell proliferation and biodegrade in a controlled fashion in parallel to cellular regeneration of cartilaginous tissue . Cartilage implants based on chondrocytes and fibrous polyglycolic acid scaffolds were recently shown to closely resemble normal cartilage histologically as well as with respect to cell density and matrix composition (collagen, glycosaminoglycan) {Freed et al., J Biomed Mater Res 27:11-23, 1993a} . These findings form the basis for developing straightforward procedures to obtain implants for clinical use from small, autologous cartilage specimens without any limitations in terms of availability of donor tissue or implant dimensions . Chondrocyte growth and cartilage matrix regeneration on polymer scaffolds are interdependent and also depend on in vitro tissue culture conditions . Under static culture conditions, cell growth rates are diffusionally limited due to increasing cell mass and decreasing effective implant porosity resulting from cartilage matrix regeneration . Optimization of the in vitro culture environment is thus essential for the cultivation of large, clinically useful cartilage implants . Preliminary studies indicate that major improvements can be achieved using bioreactors that provide efficient mass transfer and controlled shear rates at the cell and implant surfaces.

J Cell Biochem, 1993 Mar, 51(3), 252 - 6
Utilization of microgravity bioreactors for differentiation of mammalian skeletal tissue; Klement BJ et al.; Bioreactor cell and tissue culture vessels can be used to study bone development in a simulated microgravity environment . These vessels will also provide an advantageous, low maintenance culture system on space station Freedom . Although many types of cells and tissues can potentially utilize this system, our particular interest is in developing bone tissue . We have characterized an organ culture system utilizing embryonic mouse pre-metatarsal mesenchyme, documenting morphogenesis and differentiation as cartilage rods are formed, with subsequent terminal chondrocyte differentiation to hypertrophied cells . Further development to form bone tissue is achieved by supplementation of the culture medium . Research using pre-metatarsal tissue, combined with the bioreactor culture hardware, could give insight into the advantages and/or disadvantages of conditions experienced in microgravity . Studies such as these have the potential to enhance understanding of bone development and adult bone physiology, and may help define the processes of bone demineralization experienced in space and in pathological conditions here on earth.

In Vitro Cell Dev Biol, 1993 Mar, 29A(3 Pt 1), 249 - 54
Culturing of primary hepatocytes as entrapped aggregates in a packed bed bioreactor: a potential bioartificial liver; Li AP et al.; Conventional culture systems for hepatocytes generally involve cells cultured as flat, monolayer cells, with limited cell-cell contact, in a static pool of medium, unlike the liver in vivo where the parenchymal cells are cuboidal, with extensive cell-cell contact, and are continuously perfused with blood . We report here a novel bioreactor system for the culturing of primary hepatocytes with cuboidal cell shape, extensive cell-cell contact, and perfusing medium . The hepatocytes were inoculated into the bioreactor and allowed to recirculate at a rate optimal for them to collide and form aggregates . These newly-formed aggregates were subsequently entrapped in a packed bed of glass beads . The bioreactor was perfused with oxygenated nutrient medium, with controlled oxygen tension, pH, and medium perfusion rate . The hepatocytes were viable for up to the longest time point studied of 15 days in culture based on urea synthesis, albumin synthesis and cell morphology . Light microscopy studies of hepatocytes cultured for 15 days in the bioreactor showed interconnecting three-dimensional structures resembling the hepatic cell plate in the liver organ . Electron microscopy studies on the same cells revealed ultrastructure similar to the hepatocytes in vivo, including the presence of plentiful mitochondria, rough and smooth endoplasmic reticulum, glycogen granules, peroxisomes, and desmosomes . We believe that our hepatocyte bioreactor is a major improvement over conventional culture systems, with important industrial applications including toxicology, drug metabolism, and protein/peptide synthesis . The hepatocyte bioreactor concept may also be used as the basis for the development of a bioartificial liver to provide extracorporeal hepatic support to patients with hepatic failure.

Free Radic Biol Med, 1993 Mar, 14(3), 267 - 76
Hyperoxia induces DNA damage in mammalian cells; Cacciuttolo MA et al.; There is mounting evidence on the role of oxygen-derived free radicals in causing damage to various cellular components . However, most studies reported in the literature have been conducted under conditions where cells were challenged with chemical free radical generating systems . In contrast, we measured DNA strand breaks, through a relatively simple and sensitive technique, as a function of the dissolved oxygen tension in a bioreactor . Cells were exposed to a step change in oxygen tension at mid-exponential growth phase . Several levels of oxygen were tested (200, 300, and 476% dissolved oxygen with respect to air saturation at 1 atmosphere) and compared against a control (10% dissolved oxygen) . Hyperoxia was found to cause monotonically increasing DNA strand breakage at all the oxygen levels . In addition, hyperoxia was found to affect other metabolic functions such as the glucose consumption rate, lactate production rate, and cell growth . When hyperoxia-induced DNA strand breakage was compared to that induced by exposure to hydrogen peroxide, a similar response was observed . Exposure to a dissolved oxygen level of 200% induced DNA strand breakage comparable to a bolus of 4.2 microM hydrogen peroxide . Our results show that there is an association between hyperoxia and DNA damage.

Biotechnol Prog, 1993 Mar-Apr, 9(2), 131 - 7
Expansion and differentiation of human hematopoietic cells from static cultures through small-scale bioreactors; Sardonini CA et al.; Maintenance of the progenitor cells responsible for hematopoiesis has generally been accomplished using a feeder layer of stromal cells in stationary culture . Here, we compared the expansion of the total cell and progenitor cell populations using low-density mononuclear cells (LDMCs) obtained from human bone marrow in static culture (T-flasks) and in different cell culture bioreactors designed for the scale-up of mammalian cells . Static cultures were performed without the presence of a previously established stromal cell layer . Expansion of marrow in all cases was accomplished through the use of added cytokines such as IL-3, GM-CSF, and c-kit ligand . The results for the total cell expansion in static culture ranged from 4.4- to 32-fold . The cell number increase was affected by such factors as patient to patient variability, freeze-thawing, and the combination of cytokines used . Due to widespread use and the small amount of marrow needed, static cultures were used as a basis for comparison with other expansion systems . The cell culture systems used to evaluate the scale-up of marrow cultures included suspension, microcarrier, airlift, and hollow fiber bioreactors . Using identical media, cytokines, and feed schedules, LDMCs in the suspension bioreactor expanded to a value of 1.6 compared to a normalized value of 1.0 for static cultures for the two runs investigated . Expansion results for microcarrier cultures averaged 0.75 when compared to static cultures . A cell number increase in the airlift bioreactor resulted in an expansion which was 0.70 of the control static culture . Granulocyte-macrophage and erythroid progenitor assay data were also evaluated for the suspension, microcarrier, and airlift bioreactors.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotechnology (N Y), 1993 Mar, 11(3), 368 - 72
Expansion of human bone marrow progenitor cells in a high cell density continuous perfusion system; Palsson BO et al.; We describe here a continuous perfusion bioreactor system that enables a population of unselected human mononuclear bone marrow cells obtained from adult donors to expand up to 20 to 25-fold over a two-week period . Colony-forming units of granulocyte-macrophage (CFU-GM) progenitor cells expand 10 to 30-fold . These expansions depend on the gas phase oxygen concentration, the seeding density and time of cell harvest . Under operating conditions that allow for good cell proliferation, 3 to 4 million mononuclear cells can be obtained per square centimeter, with 0.5 to 0.8% being progenitor cells . Autologous human sera supported cell expansion as efficiently as animal sera . Increasing the size of the perfusion system to produce a clinically meaningful number of CFU-GMs could have important applications in bone marrow transplantation therapies.

Appl Environ Microbiol, 1993 Mar, 59(3), 682 - 6
Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences; Kane MD et al.; A fluorescently labeled version of a population-specific oligonucleotide hybridization probe was used to monitor the enrichment and isolation of a sulfate-reducing bacterium from a multispecies anaerobic bioreactor . The organism was originally identified as a molecular isolate that was phylogenetically related to Desulfovibrio vulgaris by amplification and sequencing of part of its 16S rRNA sequence . The sequence, in turn, was used to design a population-specific probe . The anaerobic medium used for the organism's enrichment and isolation was based on the physiological properties of the its closest relatives as identified by sequence comparisons . Of 30 isolates examined, only 3 hybridized with the probe . Nearly complete 16S rRNA sequences determined for each of these three isolates (i) had no mismatches with the probe target site, (ii) were identical to the amplified partial sequence of about 500 nucleotides and to one another in all other positions, and (iii) were 93.9% similar to that of D . vulgaris . In addition, one isolate chosen for further study (strain PT-2) had a substrate specificity comparable to that of D . vulgaris . These results confirmed that polymerase chain reaction amplification of 16S rRNA sequences from environmental samples can be accurate and can also provide phylogenetic information from which aspects of a population's physiology can be inferred.

Biotechnology (N Y), 1993 Mar, 11(3), 358 - 63
Expansion of primitive human hematopoietic progenitors in a perfusion bioreactor system with IL-3, IL-6, and stem cell factor; Koller MR et al.; Present methods for long-term hematopoietic culture (LTHC) employ a static culture environment which is not well-characterized . Primitive long-term culture-initiating cell (LTC-IC) numbers have been shown to decline in conventional static human LTHC, even with exogenous cytokine combinations . We have expanded human hematopoietic cells from umbilical cord blood on a preformed marrow stroma with synergistic cytokine combinations in a novel perfusion bioreactor system, which continuously maintained culture conditions within desired ranges . Interleukin-3 (IL-3) and interleukin-6 (IL-6) in perfusion culture resulted in rapid 7-day expansion of granulocyte-macrophage colony forming units (CFU-GM, 11-fold), erythroid burst-forming units (BFU-E, 2.5-fold), and granulocyte-erythroid-macrophage colony forming units (CFU-Mix, 2.4-fold), compared to 6-fold, 1.4-fold, and no expansion, respectively, in static cultures . Addition of stem cell factor (SCF) to IL-3/IL-6 in static culture increased the extent of CFU-GM expansion (to 9-fold), but did not result in BFU-E or CFU-Mix expansion . In perfusion cultures with IL-3/IL-6/SCF, much greater expansions of CFU-GM (18-fold) and CFU-Mix (5.3-fold) were obtained . More importantly, expansion of LTC-IC (nearly 3-fold in two of three experiments) was only obtained with IL-3/IL-6/SCF and perfusion . The ability to expand hematopoietic cells while maintaining or expanding primitive progenitors has potential clinical applications in bone marrow transplantation and gene therapy.

Proc Natl Acad Sci U S A, 1993 Feb 1, 90(3), 799 - 803
Rat acid phosphatase: overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties, and crystallization; Vihko P et al.; Rat prostatic acid phosphatase (rPAP; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was expressed in the baculovirus expression vector system . Recombinant protein was secreted into the medium at a high yield by infected insect cells, which were cultured at high density in a 30-liter bioreactor allowing high oxygen content for rapidly growing cells . About 20% of the cell protein produced was rPAP . Partial sequence determination of the N terminus of the purified recombinant secreted protein revealed identity to the native secreted protein, showing that the signal peptide is recognized and properly cleaved in insect cells . The enzyme was purified by using L-(+)-tartrate affinity chromatography . The purified protein had a high specific activity of 2620 mumol.min-1.mg-1 with p-nitrophenyl phosphate at the substrate, and it also showed phosphotyrosine phosphatase activity . The molecular mass of the recombinant rPAP was 155 kDa . Two subunits of 46 kDa and 48 kDa could be detected in SDS/PAGE, but only one subunit of 41 kDa was present after digestion with N-glycosidase . The active enzyme is a trimer of subunits differing only in glycosylation . When recombinant rPAP was crystallized with polyethylene glycol 6000 as the precipitant, the crystals were trigonal (space group P3(1)21) with cell dimensions a = 89.4 A and c = 152.0 A . The observed diffraction pattern extends to a resolution of at least 3 A.

Hepatology, 1993 Feb, 17(2), 258 - 65
Development of a bioartificial liver: properties and function of a hollow-fiber module inoculated with liver cells; Rozga J et al.; We have developed a bioartificial liver support system utilizing hollow-fiber bioreactor, plasmapheresis and microcarrier cell culture technologies . Liver cells were obtained through portal vein perfusion with ethylenediaminetetraacetate or ethylenediaminetetraacetate/collagenase . A mathematical model of mass transport in a hollow-fiber module, at various plasma flow velocities and system configurations, was developed . The bioartificial liver's ability to carry out specific differentiated metabolic liver functions was tested in vitro and in vivo . A reproducible large-animal model of acute ischemic liver failure was developed . Most major first-generation cyclosporine and 19-norterstosterone metabolites were isolated after substrate addition to the bioartificial liver in vitro . After bioartificial liver treatment for 6 hr (with dog or pig liver cells), dogs with acute liver failure had significantly lower serum ammonia and lactate levels and significantly higher serum glucose levels than did control animals treated with a bioartificial liver system inoculated with microcarriers alone . In addition, bioartificial liver-treated animals had significantly higher mean systolic blood pressures than did controls . Liver cell viability at the end of the 6-hr in vivo experiment was greater than 90%.

Proc Soc Exp Biol Med, 1993 Feb, 202(2), 181 - 92
Rotating-wall vessel coculture of small intestine as a prelude to tissue modeling: aspects of simulated microgravity; Goodwin TJ et al.; A new low shear stress, low turbulence microcarrier culture system has been developed at NASA's Johnson Space Center that permits large-scale three-dimensional tissue culture . Tissue culture bioreactors called rotating-wall vessels were used in conjunction with multicellular cocultivation to develop a unique in vitro tissue-modeling system . Normal small intestine epithelium and mesenchymal cells were cocultivated on Cytodex-3 microcarriers and were initiated in two phases . Normal small intestine mesenchymal cells were inoculated into the rotating-wall vessel at 2 x 10(5) cells/ml and allowed to attach and proliferate for 2 to 3 days . Normal small intestine epithelium was then added at an innoculum of 2 x 10(5) cells/ml and cultivation continued for 30 to 40 days . These cocultures attained cell numbers of 4-6 x 10(6) cells/ml and differentiated to form tissue-like masses of 0.4-0.5 cm with minimal necrosis . The masses displayed apical brush borders, differentiated epithelial cells, cellular polarity, extracellular matrix, and basal lamina . Verification of mesenchymal and epithelial cell expression was determined by immunocytochemistry and scanning electron microscopy . These data suggest that the rotating-wall vessel affords a new tissue culture model for investigation of growth, regulatory, and differentiation processes within normal tissues.

J Ind Microbiol, 1993 Feb, 12(2), 103 - 8
Effect of hydromechanical forces on the production of filamentous haemagglutinin and pertussis toxin of Bordetella pertussis; Rodriguez ME et al.; The production of Bordetella pertussis extracytoplasmic filamentous haemagglutinin (FHA) and pertussis toxin (PT) in a bioreactor under stirring conditions was studied in order to investigate the effect of hydromechanical forces on yields of both antigens . It was shown that FHA loses its haemagglutinin activity when the power transmitted by the agitator and the aerator per unit volume increases, whereas PT production is not affected . The loss of FHA activity can be explained by the action of shear forces on the filamentous structure of this antigen.

Enzyme Microb Technol, 1993 Feb, 15(2), 120 - 6
Recombinant protein production via fed-batch culture of the yeast Saccharomyces cerevisiae; Hardjito L et al.; Protein production with the recombinant yeast Saccharomyces cerevisiae in fed-batch culture is investigated in this work using beta-galactosidase as a model protein . Segregational instability was negligible during the observed culture periods . The final volumetric productivity, as determined by both cell concentration and gene expression, was strongly affected by the time course of the glucose levels in the bioreactor . It was found that an average glucose feed rate of 1.31 g glucose h-1 resulted in both the maximum beta-galactosidase production rate of 831-950 units ml-1 h-1 and the maximum cell production rate of 0.520-0.524 mg ml-1 h-1.

Adv Biochem Eng Biotechnol, 1993, 48, 53 - 77
Parameters influencing the productivity of recombinant E . coli cultivations; Friehs K et al.; In the past 10 to 15 years, many of the promises of microbial genetic engineering have been realized: the use of recombinant Escherichia coli has moved from the laboratory to the production facility, and the manufacture of therapeutic recombinant proteins such as human growth hormone and interleukins is a rapidly growing industry . Along with this progress, however, have come new problems to solve: bioreactor operators have discovered that large-scale cultivations of plasmid-containing bacteria do not behave in exactly the same way as those of plasmid-free cells, plasmid stability has been recognized as a major hurdle, and the protein product might not be present in a soluble form but rather as intracellular granules that resist solubilization . These and other difficulties represent a new generation of challenges for genetic engineering . However, genetic engineering can do more than solve these problems . Molecular biological techniques also have the ability to create new opportunities: to produce new compounds, to use cheaper substrates, to facilitate downstream processing, and to optimize production in new ways . The productivity of a cultivation can generally be expressed as the product of the cell density and the specific biological activity . Both of these parameters are influenced by a variety of factors . For recombinant cultivations, though, the level of biological activity, a reflection of the plasmid copy number and expression efficiency, is the more interesting and important consideration and will therefore be given more attention in our review . In this contribution, our general goal is to discuss the factors that influence the productivity of recombinant E . coli cultivations, covering parameters relating to DNA; parameters relating to protein synthesis; parameters relating to proteins; and parameters relating to downstream processing . The object is not to tell the reader how to choose the perfect plasmid, host, and cultivation conditions, but to make known the many variables involved in designing a recombinant process and to point out recent and potential advances made possible by genetic engineering . The discussion focuses on the production of a protein, but many of the same concepts apply to other cultivations of recombinant E . coli, including cases in which the desired product is not a protein or the cells have been designed for a special metabolic capability such as pollutant biodegradation.

NMR Biomed, 1993 Jan-Feb, 6(1), 95 - 104
Design and application of NMR-compatible bioreactor circuits for extended perfusion of high-density mammalian cell cultures; Gillies RJ et al.; MR spectroscopy of cultured cells allows non-invasive analyses of the metabolism of cells with specific phenotypes under defined conditions . This technique can be used to investigate the intracellular metabolism of cells or extended to critically evaluate phenomena observed by in vivo MRS . In this paper, a cell maintenance system is described which allows MR analyses with unparalleled spectral resolution, S/N and stability . This system consists of a 25 mm diameter hollow fiber bioreactor and a supporting circuit . The hollow fiber reactor was chosen because it yields a large filling factor which can be perfused through defined volumes . The fibers were 300 microns diameter microporous (0.2 micron) cellulose acetate/cellulose nitrate membranes with high porosity, which allow bulk convective flow throughout the extracapillary space . This flow (Starling flow) is necessary to disrupt steady-state gradients in substrates and waste products . In many respects, the design of the supporting circuit is more important than the bioreactor itself, since it provides the reactor with the proper chemical and physical environment . Hence, this circuit can be applied to a variety of bioreactor configurations . The circuit consists of a hollow fiber oxygenator and a bleed-and-feed system housed in a temperature-controlled cabinet . Culture of mammalian cells in this reactor yields 31P spectra which have excellent spectral and temporal resolution . At confluence, endogenous 31P line widths were typically < 10 Hz (at 162 MHz) and well resolved spectra were obtained in < 30 s.

Ann Biomed Eng, 1993, 21(1), 57 - 65
Bioreactor based on suspended particles of immobilized enzyme; Freed LE et al.; A bioreactor for blood detoxification was developed in which oscillation-induced secondary flows suspend particles of immobilized enzyme in a reactor operating at clinically useful flowrates . Torsional oscillation of the reactor about its axis created a pair of counterrotating toroidal vortices which were readily observed in flow-visualization studies . Oscillation frequencies were selected to provide spatially uniform particle dispersion, as assessed visually . As a model system, blood deheparinization by reactors containing heparinase immobilized to agarose particles was investigated . Identical deheparinization profiles were observed in the continuous-flow reactor and in independent batch studies, done in well mixed test tubes of blood, demonstrating that the oscillating reactor design minimizes external mass transfer limitations . Identical heparin neutralization profiles and rates were also observed in the first and the second of consecutive heparin neutralization studies (0-2 h and 2-4 h, respectively) demonstrating an effective half-life of the immobilized enzyme in the oscillating reactor of at least 4 h . No significant decrease in red or white blood cell count, platelet count, or hematocrit, and clinically acceptable levels of plasma hemoglobin and activated complement were observed with 2 h (20 passes) of in vitro recirculation of human blood through the reactor . High, stable efficacy, operational stability, and excellent biocompatibility are attributed to secondary flow induced liquid-particle mixing within the oscillating reactor.

Biomater Artif Cells Immobilization Biotechnol, 1993, 21(3), 335 - 41
Immobilized articular chondrocytes: in vitro production of extracellular matrix compounds; Ramdi H et al.; Primary cultivated rabbit articular chondrocytes were immobilized in calcium alginate beads . Both free and entrapped cells were allowed to grow under normal conditions . After bead lysis, harvested cells showed normal growth patterns when resuspended in culture medium . After long-term immobilization, the morphology and the viability of immobilized rabbit articular chondrocytes were preserved: cells remained viable and were able to grow and divide for several days inside the alginate beads in a culture incubator . The percentage of viable cells did not significantly decrease when immobilized cells were stored at 4 degrees C for 30 days . The basic metabolic properties (glucose consumption) and characteristic activities (proteoglycan secretion) were similar to those of free adherent cells with a time-dependent increase . A large scale bioproduction of extracellular matrix components may be considered of great interest for the ready-to-use complete culture systems of mammalian cells with high densities . Moreover immobilized forms also facilitate the use of cells in a bioreactor or in some unusual conditions (parabolic flights).

Bull Math Biol, 1993, 55(4), 715 - 30
An optimal time control problem for the administration of 2',3'-dideoxycytidine by using red blood cells as bioreactors; Beretta E et al.; The problem of optimal dosage is studied for the administration of ddCyd using erythrocytes as carriers and bioreactors . The volume of erythrocytes and the initial amount of drug to be loaded have to be determined in such a way that the duration of the therapeutic effect is maximized without exceeding the toxic threshold . It is found that the optimal control is unique and it is at the upper vertex of the set of the admissible controls . A more general case is also briefly discussed.

Yi Chuan Xue Bao, 1993, 20(2), 122 - 34
{Production of transgenic rabbits by micro injection}; Shi L et al.; The feasibility of using whole animal instead of bioreactor in genetic engineering has been investigated with transgenic domestic rabbits . The gene chosen is the surface gene (S gene) of hepatitis B virus . Two plasmids were specifically constructed for this purpose, pHBV3.0 contains the promoter pre S gene and a part of c gene of the virus; while MT-SA, the S gene and mouse MT promotor . These plasmids were made linear by suitable restriction endonuclease before they were transferred into maleprounclei by means of microinjection . From 757 microinjected and transplanted fertilized eggs 101 rabbits were obtained . 57% of these animals were found with integrated microinjected genes . 28 of the transgenic animals were tested for the presence of the surface antigen of the virus in the serum by ELISA method . 8 animals were found positive, approximately 30% of the tested transgenic animals . The second generation transgenics were obtained either by first generation ransgenics crossed with non-trans-genics or transgenics . Among them 73% contained the transgene and 15% had the surface antigen in the serum . Some experiments were also carried out with human growth hormone gene.

Appl Biochem Biotechnol, 1993 Spring, 39-40, 701 - 13
Biodegradation of chlorinated aliphatic hydrocarbon mixtures in a single-pass packed-bed reactor; Lackey LW et al.; Aliphatic chlorinated compounds, such as trichloroethylene (TCE) and tetrachloroethylene (PCE), are major contaminants of ground water . A single-pass packed-bed bioreactor was utilized to study the biodegradation of organic waste mixtures consisting of PCE, TCE, and other short-chain chlorinated organics . The bioreactor consisted of two 1960-mL glass columns joined in a series . One column was packed with sand containing a microbial consortia enriched from a contaminated site . The other column provided a reservoir for oxygen and a carbon source of methane/propane that was recirculated through the reactor . Sampling was accomplished by both direct headspace and liquid effluent concentration analyses . The reactor was operated in a single-pass mode . Greater than 99% degradation of trichloroethylene, approaching drinking water standards, was observed when the bioreactor residence time ranged from 1.9 to 3.2 d . Typically, when the reactor was pulse-fed with methane, propane, and air, 1 mol of TCE was degraded/110 mol of substrate utilized . Perturbation studies were performed to characterize reactor behavior . The system's degradation behavior was affected by providing different carbon sources, a pulse feeding regime, supplementing microbial biomass, and by altering flow rates.

Crit Rev Biotechnol, 1993, 13(4), 305 - 28
A review of the effects of shear and interfacial phenomena on cell viability; Hua J et al.; The shear sensitivity of animal and plant cells is a problem often encountered in large-scale cell culture . Such sensitivity varies with different cell lines and the severity of cellular damage may depend on both the magnitude and the duration of the shear stress . In a bioreactor, the shear susceptibility of cells depends on their response to hydrodynamic forces arising from fluid motions of particular scale . Cell damage may be induced by forces in the bulk liquid phase, but fluid motions associated with the gas-liquid interface are especially energetic . The detrimental effects of hydrodynamic forces are abated by the addition of some polymers, such as Pluronic F-68, methylcellulose, or serum; the exact mechanisms of protection are the subject of current research.

J Biomater Sci Polym Ed, 1993, 5(1-2), 167 - 81
Activity of horseradish peroxide adsorbed on radio frequency glow discharge-treated polymers; Chen JP et al.; Horseradish peroxidase (HRP) has been used as a model enzyme in this study of its physical adsorption and residual enzyme activity on radio frequency glow discharge (RFGD)-treated polymers . The specific enzymatic activity of HRP adsorbed on different surfaces was assumed to be an indication of the extent of its conformational alterations on the surfaces . The surfaces studied were poly (ethylene terephthalate) (PET), polytetrafluoroethylene (PTFE), and tetrachloroethylene and tetrafluoroethylene glow discharge-treated PET, abbreviated as TCE/PET and TFE/PET . All surfaces were characterized by electron spectroscopy for chemical analysis (ESCA) and liquid contact angles in air . HRP adsorbs more strongly onto the two discharge-treated surfaces than onto the untreated polymers, as evidenced by the lower amount of HRP eluted by sodium dodecyl sulfate (SDS) from the treated polymers . For example, seventy percent of the HRP adsorbed on TCE/PET or TFE/PET remains on the surface after overnight elution with a 1% solution of SDS . In contrast, untreated PET and PTFE each retains only c . 20% of the absorbed enzyme . The enzymatic activity of HRP adsorbed on the different surfaces was studied using hydrogen peroxide (H2O2) as the substrate . HRP adsorbed on the higher energy surfaces, PET and TCE/PET, retains significantly more activity than the HRP adsorbed on the lower energy surfaces, PTFE and TFE/PET which appear to destroy rapidly almost all of the activity of HRP after it adsorbs . HRP adsorbed on TCE/PET is relatively more stable over time than HRP adsorbed on PET or free HRP in solution . (For example, only 45% of the specific enzymatic activity of HRP adsorbed on TCE/PET was lost after 3 h of storage in phosphate buffer at 37 degrees C, while 70% of that adsorbed on PET was lost.) In summary, when HRP is adsorbed on TCE/PET, it is very tightly bound, and yet it maintains a significant fraction of its initial specific activity and also retains this activity for 3 h in phosphate buffer at 37 degrees C . Thus, tenacious physical adsorption of proteins such as enzymes on TCE glow discharge-treated surfaces may have potential as a new method of immobilization of such molecules, for uses in biosensors, diagnostics, bioseparations, cell culture and bioreactors.

Blood Purif, 1993, 11(3), 163 - 9
Use of sorbent columns and haemofiltration in fulminant hepatic failure; Hughes RD et al.; Artificial liver support systems were developed to remove potential toxins from the circulation of patients with liver failure . A wide range of substances have been implicated in the toxic manifestations of liver failure, but no substance(s) has been identified as being of key importance . Charcoal haemoperfusion appeared to be beneficial in early studies, but when clinical trials were performed in 137 patients at King's College Hospital there was no significant improvement in survival over intensive care alone . There have been many studies using haemodialysis/filtration systems, where an improvement in conscious level of the patient was reported, but the effect on survival was less clear . Recently in Japan, continuous high volume haemofiltration with the addition of plasma exchange has been used to support liver failure patients . In liver failure, endotoxaemia and increased cytokine production are important in the pathogenesis of circulatory failure . Experiments on adsorbents to remove cytokines have shown that Amberlite XAD-7 resin can remove significant amounts of tumour necrosis factor and interleukin-6 from liver failure plasma . The development of hepatocyte bioreactors with synthetic and metabolic function for clinical use will be a major advance in liver support.

Biomater Artif Cells Immobilization Biotechnol, 1993, 21(4), 563 - 70
Immobilization of aminoacylase in polyethyleneimine stabilized calcium alginate beads for L-phenylalanine production; Lee PM et al.; Aminoacylase I (E.C.3.5.1.14) was immobilized by entrapment in calcium alginate beads coated with polyethyleneimine for the production of L-phenylalanine by the hydrolysis of a racemic mixture of N-acetyl-DL-phenylalanine . The operational stability in terms of batch operation and continuous reaction in packed-bed bioreactor were studied . Kinetic constants, Km and Vmax values of free and immobilized enzymes were studied . Polyethyleneimine treatment was found to enhance the operational stability of the enzyme though its activity was substantially reduced . When polyethyleneimine-coated calcium alginate beads were packed into packed bed bioreactor, it was stable for at least 25 days under continuous operation without appreciable loss of activity.

Chin J Biotechnol, 1993, 9(2), 117 - 21
A domestic cell bioreactor and its application in virus culture; Dong S et al.; A cell culture bioreactor (CellCul-20) and its application in cell and virus culture are described in this paper . It has been evaluated with strict aseptic tests and one-year's operation shown that CellCul-20 bioreactor can keep its aseptic condition after being autoclaved . It can meet the requirement for the control of the main parameters for cell and virus culture and the finely adjustment of the main parameters to meet the changing conditions of the cultivation . A high cell density and a high level of virus titre were reached respectively for Vero cells and Japanese encephalitis virus (JEV) while they were cultured in this bioreactor . It is the first report on large-scale culture of JEV-infected Vero cells to prepare primary JEV vaccine . Some suggestions are made for the improvement of CellCul-20.

Chin J Biotechnol, 1993, 9(1), 19 - 26
Studies on oxygen transfer process in animal cell culture bioreactor; Wang S et al.; The oxygen transfer rates were investigated systematically in the CellCul-20A bioreactor with the device of cage aeration in this paper . The temperature, rotating speed, aeration rate and foam breaker, which affected the rate of oxygen transfer were studied, respectively . The mass transfer rate increased significantly with the aeration while the foam breaker had a negative effect on kLa, especially in a cell culture medium with 5% (v/v) calf serum . The oxygen transfer coefficients of surface and deep aerations were correlated, based on the experimental data.

Cytotechnology, 1993, 13(1), 29 - 39
The influence of dissolved oxygen tension on the metabolic activity of an immobilized hybridoma population; Thommes J et al.; In the study presented here a laboratory scale (150 ml) fluidized bed bioreactor was used as a tool for making kinetic measurements on the production of monoclonal antibodies (MAb) with a hybridoma cell line . We determined the influence of dissolved oxygen tension (DOT) on the metabolic activity of a hybridoma population immobilized in macroporous collagen microspheres . The data obtained showed a reduction of the metabolic activity of the immobilized population at reduced DOT, the total number of immobilized cells, however, remained constant . At decreasing DOT an increasing lactate yield from glucose at reduced glutamine consumption was noticed, indicating a shift in the pattern of substrate usage . A mathematical description of maintenance metabolism was formulated and the parameters of growth and maintenance requirements were calculated . A growth associated MAb production was determined under the conditions applied leading to space time yields of 225 mg MAb per liter of total reactor volume and day.

Cytotechnology, 1993, 13(2), 125 - 31
Investigation of parameters affecting a fixed bed bioreactor process for recombinant cell lines; Racher AJ et al.; A BHK 21 cell line expressing a recombinant antibody was grown in a fixed bed reactor (FBR) system using a porous support made of Siran glass beads . The contribution of five process variables (bead and inoculum sizes; circulation and dilution rates; glutamine concentration of the feed) to the productivity of the process (defined as production rate, effluent product concentration or yield of product on medium supplied) was investigated using a partial factorial experimental design . Individually, none of the variables tested had a significant affect upon productivity . The combination of smaller bead and inoculum sizes, higher circulation and dilution rates, plus higher feed glutamine concentration gave a markedly higher productivity than any other combination of variable levels tested . This combination of variable levels suggested that better results should be obtained using a fluidized bed reactor system . However, comparison of the productivities of the two systems showed that the FBR gave the better results . This result can be explained in terms of the relationship of QsrAb to mu.

Cytotechnology, 1993, 11(3), 233 - 44
Agitation, aeration and perfusion modules for cell culture bioreactors; Fenge C et al.; For an optimized bioreactor design which is adapted to the cultivation of sensitive animal cells different modular bioreactor components for gentle agitation, sufficient aeration and long-term perfusion were developed and investigated with respect to their suitability from laboratory to production scale . Aeration systems have been designed for both shear sensitive cells and cells which tolerate bubbles . The systems are based on either membranes for bubble-free aeration or stainless steel sparger systems . They were characterized by determination of their oxygen transfer capacity and optimized in cultivation processes of different cell lines under process conditions such as batch and perfusion mode . Different impellers for suspension cells and cells grown on carriers were investigated for their suitability to ensure homogeneous gentle mixing . A large pitch blade impeller as well as a novel 3-blade segment impeller are appropriate for homogeneous mixing at low shear rates . Especially with the 3-blade segment impeller fluid mechanical stress can be reduced at a given stirrer speed which is advantageous for the cultivation of cells attached to microcarriers or extremely shear sensitive suspension cells . However, our results indicate that shear sensitivity of animal cells has been generally overestimated . Continuous perfusion of both suspension cell cultures and cells cultivated on microcarriers could be successfully performed over extended periods of time using stainless steel spinfilters with appropriate pore sizes and systems based on microporous hydrophilic membranes . Spinfilters are suitable cell retention systems for technical scale bioreactors allowing continuous perfusion cultures of suspension cells (pore size 10 to 20 microns) as well as anchorage dependent cells grown on microcarriers (pore size 75 microns) over six weeks to 3 months . Applying the developed modules for agitation, aeration and perfusion process adapted bioreactor set-ups can be realized which ensure optimum growth and product formation conditions in order to maximize cell and product yields.

J Chem Technol Biotechnol, 1993, 58(1), 81 - 7
Effect of polymer additives on hydrodynamics and oxygen transfer in a bubble column bioreactor; Kawase Y; The influence of polymer additives (polyethylene oxide and polyacrylamide) on the hydrodynamics and oxygen transfer in a bubble column bioreactor was examined . The addition of small amounts of these polymers has been known to cause significant drag reduction in turbulent flow circumstances . The gas hold-up was slightly decreased and the liquid-phase mixing was somewhat enhanced due to the addition of the polymers . The addition of polymer additives brought about a reduction of the volumetric oxygen transfer coefficient by about 40% . In dilute polymer solutions, large bubbles formed by bubble coalescence moved with high rise velocities in the presence of many small bubbles and the bubble size distributions were less uniform compared with those in water . The complicated changes in bubble hydrodynamic characteristics were examined to give possible explanations for oxygen transfer reduction.

J Chem Technol Biotechnol, 1993, 57(1), 27 - 32
Immobilization of aminoacylase by encapsulation in poly-L-lysine-stabilized calcium alginate beads; Lee KH et al.; Aminoacylase I (EC 3.5.1.14) encapsulated in calcium alginate beads stabilized with poly-L-lysine was used for the production of L-phenylalanine by the hydrolysis of a racemic mixture of N-acetyl-DL-phenylalanine . The immobilized aminoacylase was studied with respect to operational stability, thermal stability, effects of pH and temperature and kinetic constants . The leakage of enzyme from the stabilized beads was eliminated . The immobilized enzyme retained high biological activity . The Km and Vmax values for the stabilized beads were 11.11 mmol dm-3 and 0.076 mumol min-1 respectively . The optimum pH and temperature for the hydrolysis were 6.5 and 55 degrees C respectively . Scanning electron micrographs revealed crosslinked structures on the surface of the beads . The operational performances of the beads in a batch reaction and a packed-bed bioreactor for continuous reaction were investigated . With batch reaction, only about 5% of enzyme activity was lost within ten reaction cycles and there was no significant loss of activity over 600 h of continuous operation after equilibrium was reached, and a conversion yield of about 80% was obtained.

J Chem Technol Biotechnol, 1993, 56(3), 233 - 9
Interaction of transport resistances with biochemical reaction in packed bed solid state fermenters: the effect of gaseous concentration gradients; Gowthaman MK et al.; Mass transfer plays an important role in solid state fermentation (SSF) systems . Earlier work on SSF in tray bioreactors indicated that steep gaseous concentration gradients developed within the substrate bed, owing to mass transfer resistances, which may adversely affect the bioreactor performance . For all practical purposes these gradients have been eliminated using a packed bed column bioreactor with forced aeration . Gaseous concentrations (oxygen and carbon dioxide) and enzyme activities were measured at various bed heights for various air flow rates during the course of fermentation . The results indicated that concentration gradients were decreased effectively by increasing air flow rate . For example, the actual oxygen and carbon dioxide concentration gradients reduced from 0.07% (v/v) cm-1 and 0.023% (v/v) cm-1 to 0.007% (v/v) cm-1 and 0.0032% (v/v) cm-1 respectively when the air flow rate was increased from 5 dm3 min-1 to 25 dm3 min-1 . This resulted in an overall improvement in the performance of the bioreactor in terms of enzyme production.

J Chem Technol Biotechnol, 1993, 56(1), 3 - 13
Designer enzyme and cell applications in industry and in environmental monitoring; Wiseman A; Renewed world interest in enzyme biotechnological industries now derives from the expectation that many new biocatalysts will be created by genetic engineering associated with protein engineering designer techniques, or by chemical modification of existing enzymes by use of protein tailoring methods . The biocatalysts produced are mainly enzymes, abzymes (catalytic antibodies) and synzymes (synthetic analogues or mimics), and these will be used in industry, synthesis, therapy: and in bioanalysis of components of foodstuffs, and the environment including water, air and soil . The biocatalysts, including whole cells, are firstly incorporated into a particular bioreactor form by use of enzyme engineering techniques such as immobilization, and are then used, as appropriate, to modify their substrates . Improved processing or enhanced products are thereby achieved in the case of manufacturing industry: or monitoring signals are generated, often in the form of a measurable change in current flow, in the case of environmental biosensors . Designer enzymes and cells can be made now for identified applications where the presently available biocatalysts are inadequate, incompatible or uncompetitive.

J Chem Technol Biotechnol, 1993, 57(1), 21 - 6
A novel approach to the production of clinical-grade dextran; Barker PE et al.; Efficient unit operations for the production and purification of the enzyme dextransucrase and the biosynthesis and fractionation of dextrans have been developed and tested . An alternative process for the production of clinical-grade dextran has been proposed by integrating these processes to achieve improved dextran product yield and the recovery of fructose as a valuable by-product . The enzyme dextransucrase, which synthesizes dextran from sucrose, has been produced either by non-aerated fed-batch or continuous fermentations, purified by continuous ultracentrifugation and ultrafiltration and used in a bioreactor-separator stage for the biosynthesis of dextran . High molecular weight dextrans have been biosynthesized on continuous counter-current chromatographic bioreactor-separator systems . Membrane filtration and size exclusion continuous chromatography were used for the fractionation of the native dextran and production of clinical-grade dextran . The advantages of the proposed process over the conventional process are related to cost reduction due to the elimination of ethanol usage and recovery, improved product quality control and the production and recovery of fructose as a by-product.

Biol Res, 1993, 26(3), 341 - 55
Oxidative metabolism and body weight: inactive, active, and mitochondrial volumes; Gunther B et al.; In homeotherms, the standardized (basal) metabolic rate should not be expressed per kilogram of body weight (specific metabolic rate), nor per unit of body surface (square meters of body-ambient interface), since both mitochondrial thermogenesis and heat-loss mechanisms (radiation, conduction, convection, evaporation) are not uniform processes . On the contrary, each organism is an heterogeneous bioreactor, which is composed at least of two compartments: 1) a metabolically active volume (aV), where oxidative phosphorylation takes place; and 2) a metabolically inactive volume (iV), where oxygen consumption is negligible . The ratio (aV/iV) is not invariant, since iV increases disproportionately with the scaling up of body size, and as shown by us, when the three main components of iV, i.e., skeleton, fat deposits, and blood volume, are added together, a similar disproportionality is found . The aV was determined by subtracting the iV from the total volume (V) of an organism, or by estimating the volume occupied by all mitochondria, or mitochondrial volume (mtV) . For this purpose two procedures are discussed: 1) the stereological or morphometric method; and 2) the oxygen consumption per unit time or physiometric method . The latter procedure is based on the equivalence between an VO2 = 3 ml O2.min-1 and a mtV of 1 ml, whose oxidative phosphorylation yields an approximate power output of 1 watt . The correspondence between oxygen consumption, heat production, and electron flux at the respiratory chain of the mitochondrial cristae, is discussed . From a physical point of view, the metabolic rate is a "power" function (P = M L2T-3), where M = mass, L = length, and T = time . The dimensional analysis and the statistical treatment of the corresponding numerical values of more than 200 allometric equations yields the 3/4 power, law established by Kleiber (1961), for the relationship between basal metabolism and body weight . Instead of expressing the metabolic rate per unit body weight (kg-1) or per unit body surface (m-2) structural and functional criteria should be taken into account as, for instance, the distinction between iV and aV, and particularly by emphasizing the paramount importance of the mtV where oxidative phosphorylation takes place . An allometric equation relating mtV and body weight (W) could be tentatively established for interspecies comparisons.

J Surg Res, 1992 Dec, 53(6), 549 - 57
Hepatocyte function in a hollow fiber bioreactor: a potential bioartificial liver; Shatford RA et al.; We have developed a novel hepatocyte loaded hollow fiber bioreactor as a potential bioartificial liver . Freshly harvested rat hepatocytes were entrapped in a three-dimensional gel matrix within hollow fibers in a perfused bioreactor . Gel entrapment allowed cells to be cultured at high density while maintaining tissue-specific function . Hepatocyte function was evaluated in 10 bioreactors, each containing approximately 5 x 10(7) cells . Oxygen consumption averaged 0.32 pmole/cell/hr, albumin appearance averaged 0.60 pg/cell/hr, and lidocaine clearance (a measure of the P-450 activity) averaged 0.74 pg/cell/hr . Function persisted for the 7 days of the study . Electron microscopy at 7 days showed the distinctive ultrastructure of viable, differentiated hepatocytes: bile canaliculi, intercellular junctions, peroxisomes, abundant mitochondria, and glycogen granules . Maintenance of tissue specific function and ultrastructure suggests that this bioreactor configuration has potential as a device to support patients in liver failure, as well as to study hepatocytes in vitro.

Int J Artif Organs, 1992 Nov, 15(11), 686 - 9
Liquid phase enzyme reactor, a new bioreactor principle; Schmer G; One of the drawbacks of solid phase enzyme reactors is the sharp decrease in substrate affinity as shown by an increase in the Km app value . To circumvent this problem a liquid phase enzyme reactor system was designed . Biotin labeled L-Tryptophan side chain oxidase (TSO) was directly injected into the plasma circuit of a filter plasmaphoresis system of rabbits and adsorbed onto an Avidin column prior to its reentry into the animal's general blood circulation . In five experiments total L-Tryptophan depletion could be observed in the plasma circuit during 60 minutes of the experiment with complete readsorption of the enzyme to the Avidin column.

J Biotechnol, 1992 Nov, 26(2-3), 257 - 73
12 beta-Hydroxylation of digitoxin by suspension-cultured Digitalis lanata cells: production of digoxin in 20-litre and 300-litre air-lift bioreactors; Kreis W et al.; A biotransformation process for the production of digoxin was developed using Digitalis lanata cell suspension cultures . Digitoxin was used as the substrate for biotransformation . Digoxin production was carried out in a variety of vessels, including 1-l exsiccators, 20-l glass reactors and a 300-l air-lift bioreactor . A culture volume of 200 l was established after 28 d and the cells were then cultured semi-continuously in a 300-l bioreactor employing the draw-fill cultivation method . Maximal digoxin production was achieved in an 8% glucose medium with a production optimum after 40-60 h of incubation in the presence of 0.65-0.8 mmol digitoxin per l . Levels of 0.52, 0.53 and 0.60 mmol digoxin per l suspension were achieved in 1-l, 20-l and 300-l vessels, respectively . About 80% of the digoxin produced was found in the bathing medium.

Appl Microbiol Biotechnol, 1992 Nov, 38(2), 141 - 6
Continuous high-cell-density fermentation of the ciliated protozoon Tetrahymena in a perfused bioreactor; Kiy T et al.; An efficient method of growing the protozoon Tetrahymena to high cell densities in a 2-1 bioreactor is described . The first phase of the fermentation is a batch phase with minimum generation times (1.4 h) . During the next phase medium is exchanged continuously using a perfusion module based on microporous hollow fibres while cell are retained . Compared to standard batch fermentations of this organism 30- to 40-fold higher cell concentrations and dry weights were achieved routinely . A maximum cell concentration of 2.2 x 10(7) cells/ml and a dry weight of 54 g/l have been obtained . As estimated from isocitrate dehydrogenase activity in the culture medium, no cell damage occurred even at high agitation rates . In addition, the cells remained viable for several weeks . Temporal limitation of the process was due to a decrease in the perfusion rate caused by blocking of the membranes . By X-ray microprobe analysis calcium phosphate depositions were detected in the pores of the clogged hollow-fibre membranes . However, even a T . pyriformis strain possessing mucocysts, dense core secretory organelles that may lead to early membrane clogging, was cultivated successfully for 3 weeks . Additionally, the consumption of nutrient protein and carbohydrates during fermentation was investigated and the effect of different perfusion rates and of glucose was studied in order to increase the efficacy of the system.

Biotechnol Prog, 1992 Nov-Dec, 8(6), 567 - 71
Recombinant beta-galactosidase production in serum-free medium by insect cells in a 14-L airlift bioreactor; King GA et al.; Spodoptera frugiperda (Sf9) insect cells were successfully cultured in serum-free medium in a 14-L airlift bioreactor . Cell densities as high as 1 x 10(7) cells/mL were achieved with specific growth rates of approximately 0.0286 h-1 (doubling time of 24 h) . This system was also used to demonstrate the expression of a reported gene, beta-galactosidase (beta-gal), when cells were infected with a recombinant baculovirus . Approximately 0.33 mg of beta-gal/mL (i.e., 104,000 units/mL) of medium were obtained at the 14-L scale, while about 0.95 mg of beta-gal/mL (i.e., 285,000 units/mL) of medium were obtained in small-scale shaker flasks . The difference was attributed to a suboptimal infection in the large scale . Specific oxygen consumption rates decreased from 5.58 x 10(-17) mol O2/cell.s in early exponential growth to 3.13 x 10(-17) mol O2/cell.s at 3 days post-infection.

Am J Obstet Gynecol, 1992 Nov, 167(5), 1470 - 8
Novel immunologic strategies in ovarian carcinoma; Freedman RS et al.; The purpose of our study was to develop new biologic systems for the treatment or diagnosis of patients with ovarian carcinoma through expansion of T-cell lines from the tumor-infiltrating lymphocytes of patients with ovarian carcinoma in low-dose recombinant interleukin-2 in sufficient numbers for treatment and human monoclonal antibodies that recognize cell-surface tumor-associated antigen determinants on ovarian carcinoma cells . Technologic advances in tumor immunology and new data presented in relation to ovarian carcinoma were used to develop T-cell lines for the treatment of advanced ovarian carcinoma patients . Logarithmic expansion of T-cell lines was performed in a hollow-fiber bioreactor, and a pilot clinical trial was initiated to treat ovarian carcinoma patients with intraperitoneal tumor-infiltrating lymphocytes plus low-dose recombinant interleukin-2 . Human hybridomas were produced by fusion of regional lymph node B cells with a heteromyeloma cell line SPATZ 4 . Two ovarian carcinoma patients have been treated with tumor-infiltrating lymphocytes expanded to 1 x 10(10) to 1 x 10(11) with manageable side effects and evidence of biologic activity . Human monoclonal antibodies have been developed that recognize tumor-associated antigen determinants . Recombinant interleukin-2-expanded tumor-infiltrating lymphocytes and human monoclonal antibodies recognize different molecular entities on tumor cells and act by different mechanisms . These approaches may be complementary to one another in future treatment strategies for ovarian carcinoma.

Biochem J, 1992 Nov 1, 287 ( Pt 3), 985 - 93
Purification and biochemical characterization of non-myristoylated recombinant pp60c-src kinase; Lydon NB et al.; To obtain sufficient material for the biochemical and biophysical study of pp60c-src, we have utilized a recombinant pp60c-src baculovirus lacking the myristoylation site at codon 2 . On infection of Sf9 cells, this virus produced large amounts of soluble non-myristoylated pp60c-src . The use of non-myristoylated pp60c-src (1) increases production of pp60c-src compared with the wild-type protein, (2) facilitates purification, (3) yields a stable product and (4) allows biochemical studies in the absence of detergents . Up to 20 mg of pp60c-src of greater than 95% purity has been purified from 6 litres of Sf9 cells grown in a bioreactor . One major and multiple minor forms of pp60c-src were separated by Mono Q f.p.l.c . Isoelectric focusing of purified pp60c-src species revealed heterogeneity, some of which could be attributed to differences in the tyrosine phosphorylation state of the enzyme . Kinetic analysis of non-myristoylated pp60c-src kinase in the presence of Mg2+ gave Km values for angiotensin II and ATP of 2 mM and 30 microM respectively and a Vmax . of 620 nmol/min per mg . The kinetic constants and metal ion preferences of a number of copolymers and peptide substrates have been compared . Polylysine and poly(GLAT), which was not phosphorylated by the pp60c-src kinase, dramatically activated autophosphorylation of Tyr-416, suggesting a conformation modulation of pp60c-src by charged polymers . This finding implies that Tyr-527 dephosphorylation is not sufficient for full activation of pp60c-src in vitro.

Ann N Y Acad Sci, 1992 Oct 13, 665, 380 - 90
The importance of cell physiology to the performance of animal cell bioreactors; Runstadler PW; This report has pointed out the following: (1) the new animal cell products that will be produced commercially will have large dollar markets and will, together with cost containment and competitive pressures, place a greater emphasis on the reduction of the cost of production through the selection of appropriate culture technology; (2) the benefits to be gained by working with basic biological processes in animal cell culture that will increase cell density, cell productivity, and product quality; (3) the need to work with reactor technologies that can affect the basic biology of the cell in these positive ways; (4) it appears worthwhile to explore immobilized, high cell density culture technologies as a possible means to achieve the objectives by affecting the basic regulation of the cell through fundamental cell/cell environment biological processes.

Ann N Y Acad Sci, 1992 Oct 13, 665, 285 - 300
Use of magnetic resonance imaging to analyze the performance of hollow-fiber bioreactors; Donoghue C et al.; Preliminary experiments were described that demonstrate that MRI is an effective tool for the noninvasive study of hollow-fiber bioreactors . Flow-compensated velocity-encoding pulse sequences were successively applied to analyze the velocity patterns in a module operated without cells, with an artificially induced flow field perturbation . Diffusion damping pulse sequences were also used to spatially resolve regions of cell growth in a bioreactor . These experiments provide the necessary basis from which future flow and spectroscopic studies can be conducted.

Enzyme Microb Technol, 1992 Oct, 14(10), 798 - 804
Effects of oxygen on recombinant protein production by suspension cultures of Spodoptera frugiperda (Sf-9) insect cells; Scott RI et al.; Spodoptera frugiperda (Sf-9) insect cells have been grown in serum-free medium in 250-ml spinner flasks . The maximum cell density obtained in these cultures was dependent on the aeration rate of the culture . Similar yields of uninfected cells were obtained when cultures were stirred in spinner flasks at 80 rev min-1 and in a 4-1 stirred-tank bioreactor and the dissolved oxygen in the bioreactor was controlled at 20% of air saturation . Cells were infected with a recombinant baculovirus at different multiplicities of infection: the timing and maximum level of expression of the recombinant protein were dependent on the multiplicity of infection, the cell density at infection, and on the aeration rate of the culture . Oxygen-limited growth resulted in undetectable levels of recombinant protein (< 6 ng recombinant protein 10(-7) cells) . Compared with the maximum yields observed in spinner flask cultures, higher levels of recombinant protein were produced when cells were grown and infected in the bioreactor . The level of dissolved oxygen in the bioreactor was controlled at 50% of air saturation.

J Biotechnol, 1992 Oct, 26(1), 83 - 97
Problems of optimisation of plant cell culture processes; Lipsky AKh; The adoption of plant cell cultures as an industrial process depends greatly on the economics of such a process . The multicycle or draw-fill culture technique is one method for improving the productivity and, hence, cost of a process . Mathematical models have been devised for the functional relationships between the nominal costs of biomass and secondary metabolites and the plant cell growth characteristics in a multicycle growth system . The models were used to evaluate the data obtained with cultures of Dioscorea deltoidea (which produces diosgenin) and Panax ginseng, grown in various types of bioreactors . The multicycle system gave an increase of 1.5-2 in biomass productivity compared with batch culture, but was probably only commercially viable if the cost of the process in the bioreactor was at least 30 times that of the medium and if an inoculum of about 30% of the culture of the previous cycle was left in the bioreactor . In the multicycle system incompletely utilised nutrient or metabolite accumulation can only reach 1.43 times or less that of the initial values . With the P . ginseng culture, about 75% of the calculated maximum cell packing density per fresh weight (approximately 530 g 1-1) in this regime was achieved . The possibility of growth in the standard bioreactor of a shear sensitive type culture was shown with a marine impeller speed up to 330 cm s-1.

J Immunol Methods, 1992 Sep 18, 154(1), 21 - 6
Selective generation of antigen-specific human hybridomas optimized for large scale growth in serum-free medium; Lang AB et al.; The direct propagation of newly formed human hybridomas in serum-free medium selects for hybrids with a metabolism best suited to growth in this environment . Under optimal culture conditions, this procedure results in the generation of antigen-specific human hybridomas comparable in frequency, stability, and antibody secretion rate to that obtained with murine hybridomas . After a transient phase of a few days in the appropriate selection medium supplemented with 1% serum, hybridomas grow in serum-free medium in stationary cultures with a cell doubling time of 15-25 h and an antibody production rate averaging 12 micrograms/10(6) cells/day . Clones propagated in bioreactors exhibited a cell doubling time of 29-35 h and an antibody secretion rate of 10-21 micrograms/10(6) cells/day.

Biochimie, 1992 Sep-Oct, 74(9-10), 931 - 9
In vivo 31P NMR study of early cellular responses to hyperosmotic shock in cultured glioma cells; Lien YH et al.; Cell volume regulation in the face of osmotic stress is a fundamental homeostatic activity, and is most critical in brain, which is spatially constrained . Despite the importance of this phenomenon, little is known about volume regulation in the brain, primarily because of the cellular heterogeneity in the tissue . We describe here simultaneous in vivo 31P nuclear magnetic resonance (NMR) measurements of cell volume, intracellular pH and phosphate metabolites during early responses to hyperosmotic stress in C6 glioma cells perfused in NMR-compatible bioreactors . Cell volume was measured using dimethyl methylphosphonate (DMMP) as a probe which has an intracellular NMR resonance shifted upfield from the extracellular resonance . The sensitivity of these measurements allowed 31P NMR spectra to be collected every 30 s . Following an increase in osmolarity from 320 to 480 mOsm by addition of NaCl to the perfusate, C6 glioma cells shrank to 67% of their original volume . We also observed a simultaneous increase of intracellular pH coincident with the decrease in cell volume . The signals from ATP decreased by 10%, but those from phosphocreatine (PCr) increased by 31% after hyperosmotic shock . However, correcting the ATP signals for the decrease in cell volume indicated that its intracellular concentrations increased after treatment . Signals from glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) were not changed significantly . This is the first in vivo report of early cellular responses monitored by NMR spectroscopy following hyperosmotic shock in cultured cells.

Int J Cell Cloning, 1992 Sep, 10(5), 299 - 308
Dynamic changes in cytokine secretion by stromal cells during prolonged maintenance under protein-free conditions; Kadouri A et al.; Stromal cells of bone marrow origin produce a variety of known cytokines and some factors exhibiting apparently new biological activities . Several of these were identified by the study of cell to cell interactions and were not found in detectable amounts in media conditioned by the cells . We describe here a culture system that enables the release of stromal cytokines into medium free of any added proteins and supplemented with peptides from casein hydrolysate (0.1%) . The absence of serum proteins allows extensive concentration and monitoring of activities that are otherwise undetectable . Stromal cells of the MBA-2.1 clonal cell line were seeded in a stationary bed reactor packed with a carrier of non-woven fabric matrix . After a proliferation phase with serum containing medium, the cells were maintained for over 10 months in protein-free medium . Throughout this extended incubation in the absence of serum or serum replacing proteins, stromal cells retained their viability and continuously released transforming growth factor-beta (TGF-beta), macrophage-colony stimulating factor (M-CSF) and restrictin-P, a cytotoxic factor that specifically arrested the growth of plasmacytoma cells . In addition, interleukin-6 (IL-6) was first undetectable, and later in culture its titer reached a maximum of 180,000 international units (IU)/ml . Concomitantly, the production of restrictin-P diminished and reached its lowest levels at the end of 10 months . The results may imply a possible causal relationship between the expression of IL-6 and restrictin-P, since no similarly significant changes were observed in the titers of M-CSF and TGF-beta . This novel bioreactor system may be adaptable for efficient production of different cytokines under absolute serum-free conditions.

Biotechnol Prog, 1992 Sep-Oct, 8(5), 462 - 4
The solution of hollow fiber bioreactor design equations; Jayaraman VK; A methodology for simplifying the solution procedure for hollow fiber bioreactor design equations has been described . Such a procedure facilitates decoupling of membrane and spongy matrix equations from the tube side equations . The equivalence between the reduced equations and the hemodialyzer problem has been explicitly obtained.

Biotechnol Prog, 1992 Sep-Oct, 8(5), 397 - 403
Fluctuations in continuous mammalian cell bioreactors with retention; Vits H et al.; Continuous flow bioreactors with cell retention have been increasingly used for the cultivation of mammalian cells . The potential advantages of such bioreactors are high cell concentrations and volumetric productivities . In many reported cases, these systems have shown fluctuations in cell concentrations of various frequency and magnitude . To analyze the dynamics of the fluctuations, a model-based approach is followed . Simulations showed that large fluctuations in biomass resulted in response to fluctuations in the retention ratio when the system is operated at high dilution rate and high cell retention . The dependence of cell concentration fluctuations on variations in dilution rate and retention ratio was established by a cross-correlation statistical analysis on available experimental data . The slower dynamics and the fluctuation propensity of retention systems suggest that continuous culture without retention is more convenient for kinetic studies . In all likelihood, continuous culture with retention can be stabilized by controlling both the retention ratio and the dilution rate.

J Biotechnol, 1992 Sep, 25(3), 289 - 306
Foaming and media surfactant effects on the cultivation of animal cells in stirred and sparged bioreactors; Zhang S et al.; Foam formation and the subsequent cell damage/losses in the foam layer were found to be the major problems affecting cell growth and monoclonal antibody (MAb) production in stirred and sparged bioreactors for both serum-supplemented and serum-free media . Surfactants in the culture media had a profound effect on cell growth by changing both the properties of bubbles and the qualities of foam formed . Comparable cell growth and MAb production in sparged bioreactors and in stirred and surface-aerated control cultures were observed only in Pluronic F-68 containing culture media . In media devoid of Pluronic F-68, cells became more sensitive to direct bubble aeration in the presence of antifoam agent which was used to suppress foam formation . Compared with serum-supplemented medium, more severe cell damage effects were observed in serum-free medium . In addition, serum-free medium devoid of cells was partially degraded under continuous air sparging . The mechanism of this damage effect was not clear . Pluronic F-68 provided protective effect to cells but not to the medium . A theoretical model based on the surface active properties of Pluronic F-68 was proposed to account for its protective effect on cell growth . Optimum media surfactant composition in terms of maximum cell growth and minimum foam formation was proposed for stirred and sparged animal cell bioreactor.

Immunobiology, 1992 Aug, 185(2-4), 207 - 21
Soluble Fc gamma R (sFc gamma R): detection in biological fluids and production of a murine recombinant sFc gamma R biologically active in vitro and in vivo; Sautes C et al.; Soluble forms of receptors for the Fc portion of IgG (sFc gamma R) were detected in biological fluids from mice and humans . In mouse bearing tumors, circulating amounts of sFc gamma R increased concurrently with tumor growth . Tumors secreting IgG2a, IgG2b or IgG3 led to a 5- to 10-fold increase in serum sFc gamma R levels whereas tumors secreting IgG1, IgGA or other types of tumors (non Ig B cell tumors, T cell lymphoma and a melanoma) increased 2- to 3-fold the levels of circulating sFc gamma R . In the human, sFc gamma R were also detected in whole unstimulated saliva . Levels of sFc gamma RII and of sFc gamma RIII were variable and did not seem to depend on the dental status of the individuals . Finally, a murine recombinant sFc gamma R (rsFc gamma R) composed of the two extracellular domains of Fc gamma RII was produced by culture of transfected L cells in bioreactors . The purified rsFc gamma R was found to inhibit antibody production in vitro in anti-SRBC responses and by cultures of small B cells stimulated by anti-IgM antibodies in the presence of IL-4 and IL-5 . Moreover, the i.p . injection of this material into adult mice immunized with SRBC led to a decrease of IgG antibody production by splenocytes, as measured by a hemolytic plaque assay, and in serum, as measured by antigen-specific ELISA.

J Cell Biochem, 1992 Aug, 49(4), 325 - 32
The prospects for domesticating milk protein genes; Hennighausen L; It is possible to convert milk glands of transgenic animals into bioreactors producing heterologous proteins such as scarce human pharmaceuticals . To predictably and successfully engineer the milk gland, we will need a thorough understanding of its physiology . Expression studies in transgenic animals have located mammary specific and hormone inducible transcription elements in the promoter/upstream regions of milk protein genes, and transfection studies in cell lines or primary cells have identified constitutive and hormone inducible elements . Most importantly, it appears that in addition to individual promoter based transcription elements structural features of milk protein chromosomal loci may contribute to the tight developmental and hormonal regulation . I will discuss milk protein gene regulation with emphasis on regulatory differences between genes and species, and the possibility that transcription elements function only properly within genetically defined chromatin domains . Novel strategies to build mammary expression vectors and to test their functionality without pursuing the standard transgenic route will be presented . Finally, I will discuss homologous recombination with the goal to target milk protein genes . Only through the domestication of milk protein genes will we be able to use their full potential in the mammary bioreactor.

Cryobiology, 1992 Aug, 29(4), 443 - 53
Effects of dimethyl sulfoxide on cultured rat hepatocytes in sandwich configuration; Rinkes IH et al.; A recently developed sandwich culture system, in which hepatocytes are sandwiched between two layers of collagen, has been shown to be capable of maintaining long-term expression of hepatocellular function (J . C . Y . Dunn et al., Biotechnol . Prog . 7, 237-245, 1991) . The development of an adequate technique for the cryopreservation of hepatocytes in such a stable culture configuration would ensure a ready supply of hepatocytes for use in bioreactors or bioartificial liver support devices . This report describes the effects of exposing hepatocytes in sandwich culture to different concentrations of the cryoprotectant dimethyl sulfoxide (Me2SO) at 22 degrees C on Day 7 of culture . Cell function, morphology, and cytoskeletal organization were followed for 14 days after exposure . Hepatocellular morphology and albumin secretion remained normal when cultures were exposed for up to 120 min to predicted final Me2SO concentrations up to 1.33 M . Exposure for less than 60 min to equilibrium concentrations of up to 3.33 M Me2SO did not adversely affect cell morphology or albumin secretion rate, but at the highest concentration (3.33 M), increase of the exposure time to 60 or 120 min resulted in dramatic, irreversible cell damage and loss of function . Actin filament organization was shown to be undisturbed when the cells were exposed to 1.33 M Me2SO for 60 min, but was irreversibly disrupted by exposure to 3.33 M for 120 min . Based on these results, a simple and safe procedure is suggested for the addition of Me2SO to hepatocytes in a sandwich culture configuration and its subsequent removal, which will be valuable for studies on hepatocyte cryopreservation.

Ann Med, 1992 Aug, 24(4), 273 - 80
Transgenic animals as bioproducers of therapeutic proteins; Janne J et al.; Many human therapeutic proteins are currently produced with the aid of recombinant DNA technology in microbial bioreactors and a few also in large-scale animal cell cultures . Although extremely cost-efficient, the microbial production system has many inherent limitations . Micro-organisms, such as bacteria, can read the universal genetic code and hence produce human proteins with correct amino acid sequence, but cannot carry out post-translational modifications, such as glycosylation, or fold the newly synthesized protein properly to ultimately generate a biologically active entity . Moreover, even though the production of the proteins as such is inexpensive, the downstream processing of the final product may be extremely difficult and costly . Many of these disadvantages, especially the lack of post-translational modifications, can be overcome by employing large-scale animal cell cultures for the production of proteins of pharmaceutical interest . However, due to the long generation time and the requirement for rich culture media, the use of animal cell bioreactors is unacceptably expensive . With the advent of transgenic technology, the production of human pharmaceuticals in large transgenic animals has become more and more attractive . The use of targeted gene transfer, the expression of the transgene of interest can be directed to occur in the mammary gland of large farm animals, such as pigs, sheep, goats or dairy cattle, and hence the transgene product is ultimately being secreted into the milk . Although not yet in commercial use, the last few years have witnessed a remarkable progress in this area and proved the feasibility of the use of 'molecular farming' in high-quantity, low-cost production of valuable therapeutic or industrial proteins . While reviewing the progress of the field over the past few years, we discuss in somewhat greater detail aspects connected with the use of dairy cattle as bioproducers of human therapeutic proteins.

J Biotechnol, 1992 Aug, 25(1-2), 145 - 82
Advanced methods for bioreactor characterization; Lubbert A; Bioreactors are characterized by the transport capacities they provide to optimally supply the microorganisms during production process . The transport is performed by flows induced in their cultivation media . In order to understand the extremely complex mixing, mass and heat transfer phenomena encountered, and to perceive their influences on bioreactor performance, sophisticated measuring techniques are required . This review compiles the developments currently in progress to surmount today's shortage of reliable measuring techniques . Measuring techniques are distinguished which can be used on different scales and their application spectra are illustrated by recently obtained results . Several new measuring techniques, which can be employed to resolve the flow structures, are discussed in detail . Only those techniques are considered which can be used to advantage during real cultivations in industrial-scale reactors.

Appl Environ Microbiol, 1992 Jul, 58(7), 2131 - 6
Limited degradation of chlorophenols by anaerobic sludge granules; Mohn WW et al.; To better understand the fate of chlorophenols treated in upflow anaerobic sludge bed reactors, we examined the ability of sludge granules from such bioreactors to degrade two trichlorophenols and one dichlorophenol in batch incubations under controlled conditions . Biodegradation was primarily limited to two distinct activities, reductive dehalogenation of ortho- and of meta-chlorine substituents . Both 3- and 4-monochlorophenol were persistent degradation products, while 2-monochlorophenol was further degraded . We also examined factors potentially affecting the rate and extent of 2,3,6-trichlorophenol degradation . An initial concentration of up to 1.75 mM (346 mg/liter) was dehalogenated . At that concentration, dehalogenation was partially inhibited but methanogenesis from formate was not . The initial concentration affected both the extent of dehalogenation and which products were detected . The maximum dechlorination rate observed was 1.4 mumol of Cl- h-1 g of volatile suspended solids-1 . Dechlorination had a temperature optimum of 50 degrees C, was inhibited by added electron acceptors, and was not appreciably affected by added electron donors . The availability of electron acceptors and electron donors did not affect the extent of chlorophenol degradation . These particular sludge granules do not appear to be capable of mineralizing phenols with meta- or para-chlorine substituents.

ASAIO J, 1992 Jul-Sep, 38(3), M468 - 72
Does a porcine hepatocyte hybrid artificial liver prolong the survival time of anhepatic rabbits?
Takahashi M, Matsue H, Matsushita M, Sato K, Nishikawa M, Koike M, Noto H, Nakajima Y, Uchino J, Komai T, et al.
To examine cultured porcine hepatocytes as a bioreactor of a hybrid artificial liver (HAL) in rabbits, a small version of a multiplated HAL was manufactured . In vitro and ex vivo studies were performed to evaluate the small HAL . The HAL consisted of 50 glass plates (10 x 5 x 0.04 cm each) on which porcine hepatocytes were cultured in a monolayer at confluent cell density . After 2 days of standard cultivation, the glass plates with hepatocytes were placed in the module and were used for studies . The module contains about 5 grams of hepatocytes (1/10 of 2 kg.bw rabbit liver) . After undergoing perfusion culture for 5 days, the HAL showed satisfactory hepatic function . Glucose and urea were synthesized at the rate of 4.06 +/- 0.7 mg/module/hr and 0.62 +/- 0.09 mg/module/hr, respectively, and loaded ammonia was metabolized at the rate of 1.29 +/- 0.26 mg/module/hr . Ex vivo extracorporeal plasma perfusion studies revealed that the module with cultured porcine hepatocytes, which were heterogeneous to rabbits, showed a tendency to prolong the survival time of anhepatic rabbits . These results indicate that the HAL system, using multiplated porcine hepatocyte monolayers, is a promising artificial liver support system for clinical cases.

Enzyme Microb Technol, 1992 Jul, 14(7), 553 - 60
Testing and evaluation of off-gas filters for bioreactors by a new bacterial aerosol challenge test method (TBAC); Kastelein J et al.; A TNO bacterial aerosol challenge (TBAC) filter test rig was developed for direct assessment of the effectiveness of bioreactor off-gas filters as an alternative to routinely applied indirect wet integrity testing (IT) . This TBAC test rig is based on bacterial aerosol challenging with Pseudomonas diminuta and dual monitoring by laser particle counting (LPC) and Andersen microbial sampling (AMS) of viable cells . The TBAC filter test rig is able to reproduce the various conditions encountered in fermentation processes . In experiments with several filters from one class, it was demonstrated that some filters were actually penetrated by up to 3,000 viable cells per test, despite their approval by commercially available IT test equipment . Repetitive filter use, prolonged use, and autoclaving of filters resulted in an increase in pressure drop over the filter but improved the performance of leaking/deviant filters due to building up of a filter cake (this phenomenon was identified by electron microscopy) . The integrity tests used were found inadequate for accurate assessment of filter quality . Certification of filter lots by random tests of commercially available off-gas filters using sensitive direct methods such as those presented here might be advisable, as not all filters purchased were of appropriate quality.

Eur J Biochem, 1992 Jul 1, 207(1), 265 - 75
Large-scale purification and characterisation of a recombinant epidermal growth-factor receptor protein-tyrosine kinase . Modulation of activity by multiple factors; McGlynn E et al.; The human epidermal-growth-factor receptor (EGF-R) is a 170-kDa transmembrane glycoprotein that mediates the mitogenic response of cells to EGF and transforming growth factor alpha . Culture conditions have been developed for the large-scale expression of the cytoplasmic domain of the EGF-R in insect cells using a recombinant baculovirus . From 61 Sf9 cells, grown to high density using a bioreactor, 20 mg of the EGF-R kinase was purified to greater than 95% purity . Purification, which was carried out in the absence of detergents using classical purification methods, yielded an EGF-R protein that was not phosphorylated on tyrosine . This procedure has enabled us to produce high quality enzyme for both structural and biochemical studies on the EGF-R kinase . The in vitro activity of the cytoplasmic domain of the EGF-R kinase was modulated by multiple assay factors which include substrates, divalent cations and conformational modulators . Kinetic analysis in the presence of Mn2+ gave an apparent Vmax value of 20 nmol min-1 mg-1 and Km values of 4.5 microM for ATP and 1.43 mM for angiotensin II . This corresponds to a turnover number of 1.4 mol min-1 mol-1 . Ammonium sulfate (1 M) resulted in an eightfold stimulation of kinase activity when assayed using angiotensin II as substrate . The specific activity of the intracellular domain of the EGF-R, when assayed at 20 degrees C in the presence of 1M ammonium sulfate, was 160 nmol min-1 mg-1 . Activation of the EGF-R kinase by ammonium sulfate was found to be substrate-specific . No activation was found when assayed using polymeric substrates . Addition of Me(2+)-ATP to the purified enzyme resulted in autophosphorylation and was accompanied by retardation of SDS/PAGE migration . Kinetic constants and metal ion preferences of a number of co-polymers and peptide substrates have been compared . Dramatic differences in kinetic constants were found which were dependent on both the substrate and metal ion used . Activation of EGF-R autophosphorylation was found to be influenced by the use of charged polymers . The random polymer of Glu, Lys, Ala, Tyr (2:5:6:1), which was not phosphorylated by the EGF-R kinase, dramatically activates autophosphorylation of the EGF-R . Thus the intracellular domain of the EGF-R appears to be in a low-activity conformation which, under appropriate assay conditions, can be activated to a similar specific activity to that reported for the purified EGF-R holoenzyme.

Appl Environ Microbiol, 1992 Jun, 58(6), 1886 - 93
Characterization of a methane-utilizing bacterium from a bacterial consortium that rapidly degrades trichloroethylene and chloroform; Alvarez-Cohen L et al.; A mixed culture of bacteria grown in a bioreactor with methane as a carbon and energy source rapidly oxidized trichloroethylene and chloroform . The most abundant organism was a crescent-shaped bacterium that bound the fluorescent oligonucleotide signature probes that specifically hybridize to serine pathway methylotrophs . The 5S rRNA from this bacterium was found to be 93.5% homologous to the Methylosinus trichosporium OB3b 5S RNA sequence . A type II methanotrophic bacterium, isolated in pure culture from the bioreactor, synthesized soluble methane monooxygenase during growth in a copper-limited medium and was also capable of rapid trichloroethylene oxidation . The bacterium contained the gene that encodes the soluble methane monooxygenase B component on an AseI restriction fragment identical in size to a restriction fragment present in AseI digests of DNA from bacteria in the mixed culture . The sequence of the 16S rRNA from the pure culture was found to be 92 and 94% homologous to the 16S rRNAs of M . trichosporium OB3b and M . sporium, respectively . Both the pure and mixed cultures oxidized naphthalene to naphthol, indicating the presence of soluble methane monooxygenase . The mixed culture also synthesized soluble methane monooxygenase, as evidenced by the presence of proteins that cross-reacted with antibodies prepared against purified soluble methane monooxygenase components from M . trichosporium OB3b on Western blots (immunoblots) . It was concluded that a type II methanotrophic bacterium phylogenetically related to Methylosinus species synthesizes soluble methane monooxygenase and is responsible for trichloroethylene oxidation in the bioreactor.

Appl Microbiol Biotechnol, 1992 Jun, 37(3), 316 - 20
A packed-bed reactor utilizing porous resin enables high density culture of hepatocytes; Yanagi K et al.; To enable high density culture of hepatocytes for use as a hybrid artificial liver support system or a bioreactor system, a packed-bed reactor using collagen-coated reticulated polyvinyl formal (PVF) resin was applied to a primary culture of hepatocytes . Cubic PVF resins (2 x 2 x 2 mm, mean pore size: 100, 250 or 500 microns) were used as supporting substrates to immobilize hepatocytes . Two hundred and fifty cubes were packed in a cylindrical column, and 2.6-11.3 x 10(7) hepatocytes were seeded in the column by irrigating with 3 ml of the medium containing hepatocytes . Perfusion culture experiments using this packed-bed reactor, as well as monolayer cultures using conventional collagen-coated petri dishes as control experiments, were performed . Sufficient amounts of hepatocytes were found to be immobilized in the reticulated structure of the PVF resins . The highest density of immobilized hepatocytes attained with PVF resin was 1.2 x 10(7) cells/cm3 PVF, which showed levels of ammonium removal and urea-N secretion comparable to those in the monolayer culture . It is concluded that the packed-bed reactor system utilizing PVF resin is a promising process for developing a bioreactor or a bioartificial organ using hepatocytes.

Enzyme Microb Technol, 1992 Jun, 14(6), 474 - 8
Electrochemical bioreactor with regeneration of NAD+ by rotating graphite disk electrode with PMS adsorbed; Miyawaki O et al.; Kinetic constants were compared among p-quinone, 2,6-dichlorophenolindophenol, phenazine methosulfate (PMS), methylene blue, and FAD in the oxidation of NADH . Among those, PMS was selected for its highest rate constant as a mediator for the electrochemical oxidation of NAD . The PMS could be stably immobilized on a graphite electrode surface by adsorption . The PMS adsorbed and that in the solution showed distinctly separated peaks in the cyclic voltammogram . The immobilized PMS functioned as an immobilized mediator to reduce the overpotential in the electrochemical oxidation of NAD so that the electrode could be used as an NAD regenerator . For the construction of an electrochemical bioreactor, a specially designed rotating disk graphite electrode was used . In spite of its extraordinarily large surface area, the behavior of the rotating disc electrode was described well by the Levich law . The NAD oxidation system of the rotating graphite disk electrode with PMS adsorbed was combined with glucose-6-phosphate dehydrogenase reaction, which reduced NAD with the consumption of glucose-6-phosphate . The electrochemical bioreactor system worked well with recycling of NAD at a high current efficiency.

J Biotechnol, 1992 Jun, 24(1), 1 - 32
A fuzzy logic controller; Yamakawa T; This paper describes a fuzzy sets method which is very useful for handling uncertainties and essential for knowledge acquisition of a human expert . Kinetics of a reactor is often complex and not trivial to describe by mathematical equations . Reactor control by traditional control technology is therefore difficult . A novel technology is presented . In the following a fuzzy inference (approximate reasoning) is used for decision making in analogy to human thinking, facilitating a more sophisticated control . Readers of this paper do not need any advanced mathematics beyond the four basic operations in arithmetic (+, -, x, divided by) and using the maximum and minimum values . This fuzzy inference is introduced to construct a fuzzy logic controller which is suitable for a nonlinear, multivariable and time variant system applied to a bioreactor.

Biotechnol Prog, 1992 May-Jun, 8(3), 204 - 10
Spin labeling and kinetic studies of a membrane-immobilized proteolytic enzyme; Zhuang P et al.; Membrane-based bioreactors can greatly influence the rate and extent of chemical reactions and consequently lower the costs associated with the corresponding engineering processes . However, in order to progress in this area, greater understanding of the relationship of the structure and function of bioreactor systems is required . In this study, a proteolytic enzyme, papain (EC 3.4.22.2), was covalently coupled onto the surface of a vinyl alcohol/vinyl butyral copolymer (PVB) membrane employing either glutaraldehyde (GA) or 1,1'-carbonyldiimidazole (CDI) . Various kinetic and performance properties of the immobilized papain were studied . It was found that these characteristics of the membrane-bound papain depended on the immobilization method . The CDI-immobilized papain bioreactor was used, although the apparent Michaelis constant, Km, of the CDI-immobilized papain was larger than that of the GA-immobilized enzyme . In separate experiments, a six-carbon spacer was also used between the membrane support and the covalently-linked enzyme . It was found that the insertion of the spacer reduced the disturbance of the enzyme system, resulting in a decreased Km, which was now closer to the value for the free enzyme . Electron paramagnetic resonance (EPR) techniques of spin labeling were used for the first time to examine the conformational change and the active site structure of an enzyme covalently immobilized to a membrane . The structural changes of the active site of papain upon immobilization with and without a spacer were in agreement with the functional properties of the enzyme.

Biotechnol Prog, 1992 May-Jun, 8(3), 244 - 51
Oxygen mass transfer enhancement via fermentor headspace pressurization; Yang JD et al.; Bioreactor headspace pressurization represents an excellent means of enhancing oxygen mass transfer to a culture . This method is particularly effective in situations where stirring or vigorous aeration is difficult . Because it in itself introduces no undesirable hydrodynamic force, the proposed method is also attractive for cells susceptible to agitation and sparging . Experiments were first conducted in an ideal fermentor by sparging air into a sulfite solution free from extraneous microbial effects . An increased oxygen mass transfer rate resulting from pressurization led to a superior cell growth rate and a higher maximum cell density in both of the microbial systems studied: a bacterial (Escherichia coli) culture up to 2.72 bar and a fragile algal (Ochromonas malhamensis) culture with pressure programming . Applying pressurization increased the maximum dry cell weight from 1.47 g/L to 1.77 g/L in the E . coli culture and increased the maximum viable cell density from 4 x 10(7) cells/mL to 10(8) cells/mL in the algal culture . An additional advantage is that formation of undesirable products under oxygen limitation, e.g., acetic acid in the E . coli culture, can be suppressed . A significant (over 250%) improvement in the oxygen transfer rate can be achieved with existing fermentors with little modification as they are already designed to withstand reasonable pressure from autoclaving . This method is simple, clean, inexpensive, and easily implemented, and it can be applied alongside other existing methods of oxygen mass transfer enhancement.

J Biotechnol, 1992 May, 23(3), 271 - 89
Optimization of a cultivation process for recombinant protein production by Escherichia coli; Yang XM; A single-stage fed-batch bioprocess for the production of a recombinant protein beta-galactosidase, by E . coli has been developed . The XL1-blue strain of E . coli which harbors a multi-number foreign plasmid PT was cultured in a reformulated medium . Critical medium components were selected and their respective concentrations were optimized with the Orthogonal Table method . An exponential substrate feeding schedule was used to maintain optimum conditions . Inhibition of growth and protein expression caused by excessive concentrations of glucose and acetate was investigated and subsequently minimized with an incremental nutrient feeding schedule which limited the specific growth rate of a culture . The program necessary to facilitate the control of substrate addition is fully described . This program has been used with a 2.5 l bioreactor and a commercially available software package for optimization without on-line or off-line measurement of optical density (OD), CO2, glucose or acetate . The optimized fed-batch process limited the acetate concentration to less than 20 mM; maintained an exponential growth phase for 50 h; and produced a cell density of 51 g l-1 dry cell weight (DCW) or 154 OD600 with a beta-galactosidase activity of 990 U ml-1.

Biotechnol Appl Biochem, 1992 Apr, 15(2), 207 - 16
Increased glucose metabolism by enzyme-loaded erythrocytes in vitro and in vivo normalization of hyperglycemia in diabetic mice; Rossi L et al.; The main metabolic properties of human red blood cells (RBC) overloaded with glucose catabolizing enzymes such as hexokinase and glucose oxidase were evaluated . Human erythrocytes loaded with human hexokinase metabolized 3.1 +/- 0.2 mumol/h/ml RBC of glucose, an amount double that consumed by normal and unloaded cells (1.46 +/- 0.16 mumol/h/ml RBC), while glucose oxidase-loaded erythrocytes consumed up to 5.5 +/- 0.5 mumol/h/ml RBC of glucose but with a time-dependent increase in methemoglobin formation due to the H2O2 produced in the glucose oxidase reaction . This methemoglobin production was greatly reduced while glucose consumption was increased (8.1 +/- 0.4 mumol/h/ml RBC) by coentrapment of hexokinase and glucose oxidase . Similar results were obtained in mouse red blood cells, although the role of hexokinase was less pronounced due to a higher basal level of this enzyme . When administered to diabetic mice the hexokinase/glucose oxidase-overloaded erythrocytes had a circulating half-life of 5 days and were able to regulate blood glucose at near physiological levels . A single intraperitoneal administration of 500 microliters of enzyme-loaded cells maintained a near-normal blood glucose concentration for 7 +/- 1 days, while repeated administrations at 10-day intervals were effective in the regulation of blood glucose levels for several weeks . These results suggest that enzyme-loaded erythrocytes can behave as circulating bioreactors and can provide a new way to reduce abnormally elevated blood glucose.

Appl Microbiol Biotechnol, 1992 Apr, 37(1), 32 - 6
Batch and continuous ribonuclease production by immobilized Aspergillus clavatus cells in a bubble-column bioreactor; Manolov RJ; Ribonuclease production using immobilized cells (IC) of Aspergillus clavatus has been studied under batch, repeated-batch and continuous fermentation conditions in a bubble-column bioreactor and compared with production by free cells . Immobilization was achieved by the method of cryostructurization in polyvinyl alcohol beads . The effect of various aeration rates in a column bioreactor has been investigated . Enzyme production by IC {42,000 units (U).1(-1)} during batch fermentations was comparable to that of a free-cell system . The specific productivity of IC was 8.5 times higher than that of free cells . In repeated batch fermentation at various aeration rates, successful reuse of IC was obtained, with comparable levels of enzyme production . Continuous ribonuclease production was achieved for 44 days at 1 vvm aeration and a dilution rate of 0.01 h-1 with high volumetric productivity (450 U.1-1.h-1) and yield.

Curr Opin Biotechnol, 1992 Apr, 3(2), 110 - 4
Mammalian cell culture processes; Hu WS et al.; Over the past year, mammalian cell culture research has been aimed at investigating the influence of culture conditions on viability, productivity and the consistency of post-translational modifications . Studies of the effect of medium conditions and the development of kinetic models are being made in relation to current efforts to develop fed-batch strategies that will optimize recombinant protein production processes . Recent advances have included novel biosensor and bioreactor developments . New technologies have also been applied to investigate high cell density bioreactor and culture conditions.

J Pathol, 1992 Apr, 166(4), 333 - 41
Lysosomes as key organelles in the pathogenesis of prion encephalopathies; Laszlo L et al.; The causation, structural origin, and mechanism of formation of spongiform lesions in transmissible encephalopathies are unknown . We have used immunogold electron microscopy to locate ubiquitin conjugates, hsp 70, and beta-glucuronidase (markers of the lysosomal compartment) and prion protein (PrP) in both control and scrapie-infected mouse brain . In scrapie-infected brain, lysosomes and lysosome-related structures (multivesicular and tubulovesicular dense bodies) are present in abnormally high numbers in neuronal cell processes . These structures contain PrP, together with the lysosomal markers ubiquitin conjugates, hsp 70, and beta-glucuronidase, which could also be identified spilling from tubulovesicular dense bodies into areas of early rarefaction in neuronal processes; we suggest that these areas of rarefaction are the precursor lesions of spongiform change . We advance the hypothesis that spongiform change is brought about by cytoskeletal disruption in neuronal processes caused by liberation of hydrolytic enzymes from lysosomes overloaded with the abnormal isoform of PrP (PrPsc) . We suggest that the lysosomal system is probably acting as the bioreactor for processing of normal PrP to the abnormal isoform . The continuous production of increasing quantities of abnormal PrPsc in lysosome-related bodies will eventually cause disruption of the lysosomal membrane with destruction of the neuronal cytoskeleton and the initiation of vacuolation . Later, death of the cell will be associated with release of the PrPsc isoform into the extracellular environment . Repeated rounds of phagocytosis, lysosomal biogenesis of PrPsc, lysosomal membrane rupture, hydrolytic enzyme release, and neuronal lysis will lead to an exponential increase in cell damage and cell death.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1992 Mar 11, 20(5), 997 - 1003
LCR/MEL: a versatile system for high-level expression of heterologous proteins in erythroid cells; Needham M et al.; We have used the human globin locus control region (LCR) to assemble an expression system capable of high-level, integration position-independent expression of heterologous genes and cDNAs in murine erythroleukaemia (MEL) cells . The cDNAs are inserted between the human beta-globin promoter and the second intron of the human beta-globin gene, and this expression cassette is then placed downstream of the LCR and transfected into MEL cells . The cDNAs are expressed at levels similar to those of the murine beta-globin in the induced MEL cells . Heterologous genomic sequences can also be expressed at similar levels when linked to to the LCR and beta-globin promoter . In addition we demonstrate that, after induction of differentiation, MEL cells are capable of secreting heterologous proteins over a prolonged time period, making this system suitable for use in continuous production systems such as hollow fibre bioreactors . The utility of the LCR/MEL cell system is demonstrated by the expression of growth hormone at high levels (greater than 100 mg/l) 7 days after induction . Since the expression levels seen do not depend upon gene amplification and are independent of the integration position of the expression cassette, it is possible to obtain clones with stable high-level expression within 3-4 weeks after transfection.

In Vitro Cell Dev Biol, 1992 Mar, 28A(3 Pt 1), 215 - 7
Culture of amebocytes in a nutrient mist bioreactor; Friberg JA et al.; We report the first use of nutrient mist bioreactor (NMB) technology to culture animal cells . The nutrient mist approximated the amebocyte stem tissue's natural environment, which is a thin layer of fluid in the gill leaflets of the horseshoe crab Limulus polyphemus . NMB culture was tried in an attempt to increase production of amebocytes, which are the source of the Limulus Amebocyte Lysate (LAL), the basis for a sensitive and commercially valuable endotoxin assay . Amebocyte growth in the nutrient mist bioreactor is comparable to growth in liquid medium . However, the current design of the bioreactor presents problems for primary cultures such as ours where a pyrogen-free environment is necessary and fungal decontamination is difficult.

Int J Artif Organs, 1992 Mar, 15(3), 162 - 7
Microcarrier culture of hepatocytes in whole plasma for use in liver support bioreactors; Cunningham JM et al.; Contact activation of plasma clotting may limit the use of some microcarrier types for hepatocyte attachment in a liver assist device . Activation by seven microcarrier types was studied in plasma containing 2-500 units heparin/mL . Clotting was activated by dextran (Cytodex 1 and 2) and collagen-coated (Cytodex 3) microcarriers at 2-25 units/mL (Cytodex 1) and 2-100 units/mL (Cytodex 2 and 3) . There was no activation by polystyrene, gelatin, glass or fibronectin-coated polystyrene microcarriers . Compared with culture medium, incubation of HepG2 cells in plasma did not affect cell viability but increased cell number (56.4 versus 65.1 x 10(4) cells; P less than 0.05) and incorporation of {3H}-amino acids into protein (204913 versus 279624 dpm; P less than 0.05) . Polystyrene-attached cells demonstrated time-linear protein synthesis, glucose and 7-ethoxycoumarin metabolism . We conclude that polystyrene-attached hepatocytes maintain viability and metabolic activity in plasma and are of potential use in a liver support bioreactor.

Can J Microbiol, 1992 Mar, 38(3), 222 - 5
Microcarrier culture of fish cells and viruses in cell culture bioreactor; Chen Z et al.; This article deals with the culture of grass carp (Ctenopharyngodon idellus) lip and embryo cells on Cytodex 3 and GT-2 microcarriers in a 1.5-L cell culture bioreactor to propagate grass carp hemorrhage virus . The cells and viruses were successfully cultivated at 26 degrees C, pH 7.0, and dissolved oxygen 40% of air saturation . The cell density achieved was as high as 7.4 x 10(6) cells/mL, and the virus titre reached 6.75 log LD50/0.5 mL from an initial 3.00 log LD50/0.5 mL . The results present broad prospects for fish virus vaccine production.

Enzyme Microb Technol, 1992 Mar, 14(3), 203 - 8
Cultivation of mammalian cells on macroporous microcarriers; Nikolai TJ et al.; Adherent cells can be cultivated in a stirred-tank bioreactor by attaching to microcarriers . Macroporous microcarriers, with their intraparticle space and surface area for cell growth, can potentially support a higher cell concentration than conventional microcarriers, which support cell growth only on the external surface . Chinese hamster ovary (CHO) cells and green monkey kidney (Vero) cells were cultivated on macroporous microcarriers, Cultispher-G . Cells attached to the microcarriers at a slow rate and grew to a high density . Thin sections of the microcarriers demonstrate that cells were initially on the exterior of the microcarriers and migrated into the interior as cell concentration increased . Vero cells cultivated on these microcarriers were successfully used for the production of vesicular stomatitis virus (VSV).

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