Int J Artif Organs, 2002 Oct, 25(10), 1001 - 5 Primary human liver cells as source for modular extracorporeal liver support--a preliminary report; Sauer IM et al.; Cell-based extracorporeal liver support is an option to assist or replace the failing organ until regeneration or until transplantation can be performed . The use of porcine cells or tumor cell lines is controversial . Primary human liver cells, obtained from explanted organs found to be unsuitable for transplantation, are a desirable cell source as they perform human metabolism and regulation . The Modular Extracorporeal Liver Support (MELS) concept combines different extracorporeal therapy units, tailored to suit the individual and intra-individual clinical needs of the patient . A multi-compartment bioreactor (CellModule) is loaded with human liver cells obtained by 5-step collagenase liver perfusion . A cell mass of 400 g - 600 g enables the clinical application of a liver lobe equivalent hybrid organ . A detoxification module enables single pass albumin-dialysis via a standard high-flux dialysis filter, and continuous veno-venuous hemodiafiltration may be included if required . Cells from 54 human livers have been isolated (donor age: 56 +/- 13 years, liver weight: 1862 +/- 556 g resulting in a viability of 55.0 +/- 15.9%) . These grafts were not suitable for LTx, due to steatosis (54%), cirrhosis (15%), fibrosis (9%), and other reasons (22%) . Out of 36 prepared bioreactors, 10 were clinically used to treat 8 patients with liver failure . The overall treatment time was 7-144 hours . No adverse events were observed . Initial clinical applications of the bioreactor evidenced the technical feasibility and safety of the system. Int J Artif Organs, 2002 Oct, 25(10), 994 - 1000 Cultivation of porcine hepatocytes in polyurethane nonwovens as part of a biohybrid liver support system; Linti C et al.; Many patients suffering from end-stage liver disease cannot be transplanted within reasonable time due to the shortage of donor organs . Bioartificial liver support systems may contribute to the liver regeneration or bridging the time until a liver graft for transplantation becomes available . Nonwovens with integrated oxygenation capacity have been developed and manufactured by melt blow technology using thermoplastic polyurethane . Capillary membranes for oxygenation were integrated into the nonwoven during the processing . The polyurethane nonwoven structures with adapted pore size and high pore volume allow high cell densities in the hepatocyte culture . The three-dimensional cell culture was housed by a flow bioreactor system and was integrated in a closed loop circulation with monitoring possibilities for pressure, pH, temperature, ammonia, and oxygen . Hepatocytes were isolated from rats or pigs by collagenase perfusion and infused into the medium-perfused circulation . Cells showed high viability and hepatocyte specific cytochrome P450-dependent metabolic function in culture (MEGX test). Int J Artif Organs, 2002 Oct, 25(10), 975 - 84 A new bioassay including a small scale hepatocyte bioreactor for hepato-mediated toxicity testing in a target cell line; Deglmann CJ et al.; New approaches for in vitro testing of hepato-mediated toxicity are undertaken to offer alternatives to in vivo animal testing . The described bioassay for hepato-mediated toxicity testing is based on a small scale hepatocyte-bioreactor with pig hepatocytes connected to a silicon sensor based microphysiometer system for monitoring of the extracellular acidification rate (EAR) of cells and the microphysiometer alone . EAR represents the metabolic activity of tested cells (hepatocytes and ZR 751 cells) under the influence of perfused media, compared to controls, which were set to 100% . Cyclophosphamide (CYCL), whose cytostatic effect is dependent on CYP 450 biotransformation was used as a model substrate . CYCL showed decrease of EAR in hepatocytes, but not in ZR 751 cells . Bioreactor supernatant including CYCL was pumped into the microphysiometer and EARs of the target ZR 751 cell line were recorded . After 7 h of bioreactor supernatant perfusion the ZR 751 cell line showed an EAR decrease of 18.68% +/- 10.18, as compared to controls (bioreactor supernatant from the identical set-up without CYCL) . Thus the presented model of hepato-activated toxicity showed an EAR decrease in the ZR 751 cell line that reflected the toxic activation of CYCL by the bioreactor . This new bioassay serves as an example of future applications for hepatocyte bioreactors in automated toxicity testing devices, e.g . in preclinical drug studies or evaluation of hepato-mediated toxicity, not depending on cell destruction or further assays. Int J Artif Organs, 2002 Oct, 25(10), 966 - 74 Treatment of acute liver failure in pigs reduces hepatocyte function in a bioartificial liver support system; Abrahamse SL et al.; Several different types of bioartificial liver (BAL) support systems have been developed to bridge patients suffering from acute liver failure (ALF) to transplantation or liver regeneration . In this study we assessed the effects of ALF plasma on hepatocyte function in the BAL system that has been developed in our center . Pigs (40-60 kg) were anaesthetised and a total hepatectomy was performed . Cells were isolated from the resected livers and were transferred to the bioreactor of the BAL system . Twenty hours after cell isolation, hepatocytes in the BAL were tested for cell viability and functional activity by using a recirculating test medium in which assessment of LDH leakage, ammonia clearance, urea synthesis, 7-ethoxycoumarin O-deethylase (ECOD) activity and pseudocholine esterase production was performed . Subsequently, two groups were studied . In one group (I, n=5), the cell-loaded bioreactor was used to treat the donor pig, rendered anhepatic, for 24 hours . In the second group (II, n=5) the bioreactor was cultured for 24 h and served as a control . After 24 hours treatment or culturing, the cell viability count and functional activity tests were repeated . The results show that hepatocytes in the BAL remained viable after 24 h treatment of anhepatic pigs, as shown by the LDH release and pseudocholine esterase production . However, metabolic functions such as ammonia clearance, ECOD and urea synthesis were reduced after 24 h exposure of hepatocytes to autologous ALF plasma, whereas these functions were unaltered after 24 h culturing of the cells in the bioreactor. Int J Artif Organs, 2002 Oct, 25(10), 960 - 5 ALEX (artificial liver for extracorporeal xenoassistance): a new bioreactor containing a porcine autologous biomatrix as hepatocyte support . Preliminary results in an ex vivo experimental model; Ambrosino G et al.; Long-term maintenance of viability and expression of differentiated hepatocyte function is crucial for bioartificial liver support . We developed a new bioreactor design (ALEX), associated with a new extracellular autologous hepatocyte biomatrix (Porcine Autologous Biomatrix - PBM) support . To test this new bioreactor, we compared it to a standard BAL (BioArtificial Liver) cartridge in a ex vivo model using human plasma added to bilirubin, ammonium and lidocaine . A pathology study was performed on both bioreactors . The results suggest that ALEX allows a maximal contact between the perfusing plasma and the liver cells and a proper hepatocyte support by a cell-to-matrix attachment . ALEX is a suitable cell support bioreactor, guaranteeing long-term maintenance of the metabolic activity of hepatocytes when compared to a standard BAL cartridge. Int J Artif Organs, 2002 Oct, 25(10), 939 - 49 Advanced technology for extracorporeal liver support system devices; Borra M et al.; Acute Liver Failure (ALF) still presents high mortality rates, and liver transplant is the only treatment with proven efficacy . However transplant is not always possible and systems for Extracorporeal Liver Support (ELS) are being developed which can treat patients with ALF, for whom a transplant is not available, or is delayed . They can also treat patients with chronic liver disease who develop ALF . There are two types of ELS: artificial systems (hemoperfusion, plasmaperfusion, therapeutic plasma exchange, continuous hemodialysis and high volume continuous hemofiltration) and bioartificial systems . These are based on a biological component (animal or human hepatocytes) inserted into a bioreactor, whose main function is to perform the metabolic activity and synthesis that the liver can no longer perform . The results obtained in clinical trials have so far shown that the best results in terms of compensating for lost metabolic function and detoxification are obtained inserting artificial components in the bioartificial circuit. ASAIO J, 2002 Nov-Dec, 48(6), 592 - 7 Development of a new bioartificial liver using a porcine autologous biomatrix as hepatocyte support; Ambrosino G et al.; Long-term maintenance of hepatocyte viability and differentiated function expression is crucial for bioartificial liver support . The maintenance of hepatocyte function in a bioreactor is still a problem . A major advance was the recognition that hepatocytes in attachment cultures can maintain their differentiation longer . To restore hepatocyte polarity and prolong their function, we developed a new bioreactor with a cross-flow geometry configuration and an original hepatocyte extracellular autologous biomatrix (Porcine Bio-Matrix) support . To test this new bioreactor, we compared it with a standard bioartificial liver cartridge in a suitable surgical model of acute liver failure in pigs . In our model, we performed a total hepatectomy, followed by partial liver transplantation after an 18 hour anhepatic phase . The results showed that the bioreactor containing the biomatrix was able to bridge the animal to transplantation and to sustain the transplanted liver until all function recovered (80% of animals survived, p = 0.0027) . No animal survived more than 24 hours after liver transplantation in the group treated with the traditional bioartificial liver, whereas hepatocyte viability on the Porcine Bio-Matrix was 65% after 12 hours of treatment . The results suggest that our biomatrix is a suitable cell support and guarantees long-term maintenance of metabolic activity of hepatocytes . Further studies are needed, but the results obtained with this new three-dimensional bioreactor are promising, and its potential is attractive. Biorheology, 2003, 40(1-3), 331 - 6 Development and validation of a bioreactor for physical stimulation of engineered cartilage; Demarteau O et al.; A bioreactor has been developed to apply different regimes of physical stimulation to tissue specimens under highly controlled conditions . The computer-controlled device exposes specimens to compressive deformation at various strains and frequencies, measures the load applied to each sample and allows simultaneous medium stirring at different velocities . Validation tests confirmed the accuracy of the system in (i) its displacement (errors averaged 0.072+/-0.051 microm), and in (ii) setting the contact with the samples utilizing micrometer screws coupled to plungers (errors averaged 1.74+/-0.36% for samples of 1.60-3.18 mm thickness), thus ensuring accurate compressive deformation . The developed bioreactor, which represents an advance in the technology for physical stimulation of tissue specimens, is currently used to apply compressive deformation and hydrodynamic forces to human chondrocytes cultured in biodegradable polymer scaffolds, with the goals of (i) engineering functional grafts for the repair of cartilage defects (ii). Biotechnol Bioeng, 2003 Jan 20, 81(2), 193 - 210 Enclosed outdoor photobioreactors: light regime, photosynthetic efficiency, scale-up, and future prospects; Janssen M et al.; Enclosed outdoor photobioreactors need to be developed and designed for large-scale production of phototrophic microorganisms . Both light regime and photosynthetic efficiency were analyzed in characteristic examples of state-of-the-art pilot-scale photobioreactors . In this study it is shown that productivity of photobioreactors is determined by the light regime inside the bioreactors . In addition to light regime, oxygen accumulation and shear stress limit productivity in certain designs . In short light-path systems, high efficiencies, 10% to 20% based on photosynthetic active radiation (PAR 400 to 700 nm), can be reached at high biomass concentrations (>5 kg {dry weight} m(-3)) . It is demonstrated, however, that these and other photobioreactor designs are poorly scalable (maximal unit size 0.1 to 10 m(3)), and/or not applicable for cultivation of monocultures . This is why a new photobioreactor design is proposed in which light capture is physically separated from photoautotrophic cultivation . This system can possibly be scaled to larger unit sizes, 10 to >100 m(3), and the reactor liquid as a whole is mixed and aerated . It is deduced that high photosynthetic efficiencies, 15% on a PAR-basis, can be achieved . Future designs from optical engineers should be used to collect, concentrate, and transport sunlight, followed by redistribution in a large-scale photobioreactor . Biotechnol Bioeng, 2003 Jan 20, 81(2), 178 - 86 Characterization of gas-liquid mass transfer phenomena in microtiter plates; Hermann R et al.; Gas-liquid mass transfer properties of shaken 96-well microtiter plates were characterized using a recently described method . The maximum oxygen transfer capacity (OTR(max)), the specific mass transfer area (a), and the mass transfer coefficient (k(L)) in a single well were determined at different shaking intensities (different shaking frequencies and shaking diameters at constant filling volume) and different filling volumes by means of sulfite oxidation as a chemical model system . The shape (round and square cross-sections) and the size (up to 2 mL maximum filling volume) of a microtiter plate well were also considered as influencing parameters . To get an indication of the hydrodynamic behavior of the liquid phase in a well, images were taken during shaking and the liquid height derived as a characteristic parameter . The investigations revealed that the OTR(max) is predominantly dependent on the specific mass transfer area (a) for the considered conditions in round-shaped wells . The mass transfer coefficient (k(L)) in round-shaped wells remains at a nearly constant value of about 0.2 m/h for all shaking intensities, thus within the range reported in the literature for surface-aerated bioreactors . The OTR(max) in round-shaped wells is strongly influenced by the interfacial tension, determined by the surface tension of the medium used and the surface properties of the well material . Up to a specific shaking intensity the liquid surface in the wells remains horizontal and no liquid movement can be observed . This critical shaking intensity must be exceeded to overcome the surface tension and, thus, to increase the liquid height and enlarge the specific mass transfer area . This behavior is solely specific to microtiter plates and has not yet been observed for larger shaking bioreactors such as shaking flasks . In square-shaped microtiter plate wells the corners act as baffles and cause a significant increase of OTR(max), a, and k(L) . An OTR(max) of up to 0.15 mol/L/h can be reached in square-shaped wells . Biodegradation, 2002, 13(2), 155 - 62 Biodegradation of VOCs from printing press air by an on-site pilot plant bioscrubber and laboratory scale continuous yeast cultures; Granstrom T et al.; The volatile organic compound composition (VOCs) of printing press air was found to contain mostly ethanol, but also ethyl acetate, 1-propanol, 2-propanol, 1-methoxy-2-propanol and 3-ethoxy-1-propanol . A pilot plant bioscrubber inoculated with a mixed microbial population was constructed on-site . The bioscrubber was able to treat the polluted gas efficiently . It, however, suffered from strong wall growth and blockages in the column . The efficiencies of the pilot plant and a bioreactor is compared . The yeasts Candida guilliermondii and Saccharomyces cerevisiae known to tolerate ethanol were selected instead of mixed population to avoid the wall growth a nd blockages inthe bioreactor . The removal of the VOCs both individually and as a complex mixture was tested in a microcultivation system and in continuous chemostat cultures with and without cell recycling . The Candida yeast could use all the compounds as a carbon source while growth of S . cerevisiae was markedly slower on the methoxylated and ethoxylated propanols . Best total removal of the VOCs was 99% and achieved by C . guilliermondii . The only compound that was not totally removable in the chemostat experiment with C . guilliermondii was 1-methoxy-2-propanol . In laboratory scale the total and volumetric removal of VOCs by C . guilliermondii was more efficient compared to the pilot plant encouraging to scale up and applying the yeast bioreactor to real field conditions. Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 693 - 702 The anammox case-a new experimental manifesto for microbiological eco-physiology; Strous M et al.; The anaerobic ammonium oxidation process is a new process for ammonia removal from wastewater . It is also a new microbial physiology that was previously believed to be impossible . The identification of Candidatus Brocadia anammoxidans and its relatives as the responsible bacteria was only possible with the development of a new experimental approach . That approach is the focus of this paper . The approach is a modernisation of the Winogradsky/Beyerinck strategy of selective enrichment and is based on the introduction of the molecular toolbox and modern bioreactor engineering to microbial ecology . It consists of five steps: (1) postulation of an ecological niche based on thermodynamic considerations and macro-ecological field data; (2) engineering of this niche into a laboratory bioreactor for enrichment culture; (3) black-box physiological characterisation of the enrichment culture as a whole; (4) phylogenetic characterisation of the enriched community using molecular tools; (5) physical separation of the dominant members of the enrichment culture using gradient centrifugation and the identification of the species of interest in accordance with Koch's postulates; (6) verification of the in situ importance of these species in the actual ecosystems . The power of this approach is illustrated with a case study: the identification of the planctomycetes responsible for anaerobic ammonium oxidation . We argue that this was impossible using molecular ecology or conventional 'cultivation based techniques' alone . We suggest that the approach might also be used for the microbiological study of many interesting microbes such as anaerobic methane oxidisers. Water Sci Technol, 2002, 46(9), 201 - 9 High salinity wastewater treatment using yeast and bacterial membrane bioreactors; Dan NP et al.; Two laboratory-scale membrane bioreactor systems were investigated to treat high salinity wastewater containing high organic (5,000 mg/L COD) and salt content (32 g/L NaCl), namely: (1) the Yeast Membrane Bioreactor (YMBR) and; (2) Yeast pretreatment followed by Bacterial Membrane Bioreactor (BMBR) . In the YMBR system, experimental runs were conducted with a mean biomass concentration of 12 g MLSS/L . Here the maximum COD removal rate of 0.93 g COD/g MLSS x day was obtained at F/M of 1.5 g COD/g MLSS.d . Whereas, the BMBR system was operated with a biomass concentration of up to 25 g MLSS/L, resulting in maximum COD removal rate of 0.32 kg COD/kg MLSS x day at F/M ratio of 0.4 . In comparison to BMBR, YMBR could obtain higher COD removal rate at higher organic loading, indicating the potential of a yeast reactor system to treat high salinity wastewater containing high organic concentration . Transmembrane pressure in BMBR was progressively increased from 2 to 60 kPa after 12 d, 6 d and 2 d at a hydraulic retention time (HRT) of 14 h, 9 h and 4 h, with average biomass concentration of 6.1, 15 and 20 g MLSS/L, respectively . Whereas the transmembrane pressure in YMBR has increased from 2 to 60 kPa only after 76 days of operation, with an average biomass concentration of 12 MLSS/L and an operating HRT range of 5-32 h. Water Sci Technol, 2002, 46(9), 193 - 200 The effect of aeration and non-aeration time on simultaneous organic, nitrogen and phosphorus removal using an intermittent aeration membrane bioreactor; Ujang Z et al.; A laboratory-scale membrane bioreactor (MBR) was fed with synthetic wastewater to investigate the possibility of simultaneous removal of organic, nitrogen and phosphorus by intermittent aeration . The MBR consists of two compartments using a microfiltration membrane with 0.2 microm pore size and a surface area of 0.35 m2 . Hydraulic retention time was set at 24 hours and solid retention time 25 days . MLSS concentration in the reactor was in the range of 2,500-3,800 mg/L . The MLSS internal recycling ratio was maintained at 100% influent flow rate . Intermittent aeration was applied in this study to provide an aerobic-anaerobic cycle . Three stages of operations were conducted to investigate the effect of aeration and non-aeration on simultaneous organic and nutrient removal . In Stage 1, time cycles of aeration and non-aeration were set at 90/150 min and 150/90 min in the first and second compartment, the removal efficiency was 97%, 94% and 70% for COD, nitrogen and phosphorus respectively . In Stage 2, time cycles of aeration and non-aeration were set at 60/120 min and 120/60 min in the first and second compartment, the removal efficiency was 97%, 96% and 71% for COD, nitrogen and phosphorus respectively . In Stage 3, time cycles of aeration and non-aeration were set at 120/120 min and 120/120 min in compartment 1 and 2, the removal efficiency was 98%, 96% and 78% for COD, nitrogen and phosphorus respectively . Results show that longer non-aeration time in the second compartment provided better performances of biological phosphorus removal. J Physiol, 2002 Nov 15, 545(Pt 1), 199 - 206 ATP release from human airway epithelial cells studied using a capillary cell culture system; Guyot A et al.; Epithelial release of adenosine triphosphate (ATP), an important autocrine and paracrine signalling molecule, is acutely mechanosensitive and therefore difficult to study . We describe here a novel preparation that minimizes mechanical and metabolic perturbations, and use it to examine ATP secretion by epithelial cells . The Calu-3 cell line derived from human airway sub-mucosal glands was cultured in a hollow fibre bioreactor on porous capillaries that were perfused by a re-circulating medium pump . Cells became polarized and cultures were stable for > 5 months, as evidenced by microscopy and lactate production (approximately 250 microg (10(8) cells)(-1) day(-1)) . Elevating apical flow rate 5-fold increased ATP secretion from approximately 200 to 6618 fmol min(-1) . Reducing apical osmolarity by 25-43 % also increased ATP secretion, which then declined spontaneously to a plateau rate that persisted as long as hypotonic perfusion was maintained . Release deactivated rapidly after shear and osmotic stresses were terminated, and was not associated with detectable cell lysis . Lowering apical {Ca(2+)} to increase connexin hemichannel permeability also stimulated ATP release and increased secretion during both hyposmotic and shear stress; however, the connexin 43 blocker flufenamic acid inhibited shear-induced ATP release only in low-Ca(2+) solution, and therefore another secretory pathway may operate with physiological (i.e . mM) calcium . Regardless of the mechanism, the present results quantify ATP responses to mechanical and osmotic stimuli and demonstrate the usefulness of capillary cultures for studying epithelial secretion. J Microencapsul, 2002 Sep-Oct, 19(5), 571 - 90 Quantitative study of the production and properties of alginate/poly-L-lysine microcapsules; Gugerli R et al.; Alginate-polylysine-alginate (APA) microcapsules are of particular interest for their application as implants or for bioreactor cultures . Although their formation has been widely studied, there is still a lack of quantitative data describing resistance, membrane thickness and permeability . In this study, the quantitative application of a Texture Analyser for the measurement of capsule deformation yielded important results that permit comparison with other polymer systems used for encapsulation . Furthermore, single-membrane and multi-membrane capsules were formed in order to improve the modulation of the capsule properties . For single-membrane capsules, resistance was mostly affected by the incubation time in poly-L-lysine (PLL), the PLL molecular weight and concentration . The increase in resistance from 0.1 +/- 0.01 g/capsules to 2 +/- 0.2 g/capsules was linked to a membrane thickening (35-120 microm) and a decrease in permeability (150 to 40 kD) . Thus, it was not possible to modify resistance and membrane permeability independently . Multi-membrane capsules with a resistance comparable to single-membrane capsules could be formed using various combinations of PLL molecular weights, and enabled uncoupling of permeability and resistance properties. Anal Chem, 2002 Nov 1, 74(21), 5507 - 12 Mass spectrometric immunoassay for parathyroid hormone-related protein; Lu CM et al.; This paper describes a novel two-site peptide immunoassay using the isotope 14C as the label and accelerator mass spectrometry as the detection system . A mouse monoclonal antibody (1A5) against the amino terminal region of human parathyroid hormone-related protein (PTHrP) was labeled with 14C by growing the hybridoma cells in a miniPERM bioreactor in the presence of {U-14C}L-leucine and {U-14C}D-glucose . The antibody was purified from the culture media using protein G affinity chromatography . The purified 14C-labeled antibody (14C-1A5) fractions showed excellent correlation between the levels of radioactivity and binding activity for PTHrP . Using 14C-1A5 as the detection antibody in a two-site immunoassay format for PTHrP1-141, a 16-kDa polypeptide, an analytic sensitivity of 10 pmol/L was achieved with a linear measurement range up to 1.3 nmol/L . Only approximately 17 pCi/ well (or 1.6 nCi/96-well microtiter plate) 14C-1A5 was used, which is far below the limit (50 nCi/g) for disposal as nonradioactive waste . This study may serve as a model for the development of sensitive and "nonradioactive" immunoassays for peptides, including polypeptide tumor markers. Biotechnol Bioeng, 2003 Jan 5, 81(1), 106 - 14 The response of virally infected insect cells to dissolved oxygen concentration: recombinant protein production and oxidative damage; Saarinen MA et al.; The effects of dissolved oxygen (DO) concentration on virally infected insect cells were investigated in 3-L bioreactor culture . Specifically, cultures of Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) were infected with Autographa californica multiple nucleopolyhedrovirus expressing secreted alkaline phosphatase (SEAP) . Following infection at a DO concentration of 50% air saturation, the DO concentration was adjusted to a final value of either 190%, 50%, or 10% air saturation . Recombinant SEAP production, cell viability, protein carbonyl content, and thiobarbituric acid reactive substances (TBARS) content were monitored . The increases in protein carbonyl and TBARS contents are taken to be indicators of protein oxidation and lipid oxidation, respectively . DO concentration was found to have no noticeable effect on SEAP production or cell viability decline in the Sf-9 cell line . In the Tn-5B1-4 cell line, cells displayed an increased peak SEAP production rate for 190% air saturation and displayed an increased rate of viability decline at increased DO concentration . Protein carbonyl content showed no significant increase in the Sf-9 cell line by 72 h postinfection (pi) at any DO concentration but showed a twofold increase at 10% and 50% DO concentration and a threefold increase at 190% DO concentration by 72 h pi in Tn-5B1-4 cells . TBARS content was found to increase by approximately 50% in Sf-9 cells and by approximately twofold in Tn-5B1-4 cells by 72 h pi with no clear relationship to DO concentration . It is hypothesized that oxygen uptake changes due to the viral infection process may bear a relation to the observed increases in protein and lipid oxidation and that lipid oxidation may play an important role in the death of virally infected insect cells . Biotechnol Bioeng, 2003 Jan 5, 81(1), 80 - 91 Unveiling steady-state multiplicity in hybridoma cultures: the cybernetic approach; Namjoshi AA et al.; Mammalian cells grown in suspension produce waste metabolites such as lactate, alanine, and ammonia, which reduce the yield of cell mass and the desired product on the nutrients supplied . Previous studies (Cruz et al., 1999; Europa et al., 2000; Follstad et al., 1999) have shown that the cells can be made to alter their metabolism by starving them on their nutrients in continuous cultures at low dilution rates or starting the culture as a fed-batch . This leads to multiple steady states in continuous reactors, with some states being more favorable than others . Mathematical models that take into account the metabolic regulation that leads to these multiple steady states are invaluable tools for bioreactor control . In this article we present a cybernetic modeling strategy in which Metabolic Flux Analysis (MFA) is used to guide the cybernetic formulation . The hybridoma model presented as a result of this strategy considers the partially substitutable, partially complementary nature of glucose and glutamine . The choice of competitions within the network is guided by MFA and the model is successful in explaining the three multiple steady states observed . The cybernetic model though identified for the hybridoma experiments of Hu and others (Europa et al., 2000) seem generally applicable to mammalian systems as it captures the pathways that are common to mammalian cells grown in suspension . The model presented here could be used for start-up strategies for continuous reactors and model-based feedback control for maintaining high productivity of the reactor . Biotechnol Bioeng, 2003 Jan 5, 81(1), 66 - 73 Distributed model of solid waste anaerobic digestion: effects of leachate recirculation and pH adjustment; Vavilin VA et al.; A distributed model of solid waste digestion in a 1-D bioreactor with leachate recirculation and pH adjustment was developed to analyze the balance between the rates of polymer hydrolysis/acidogenesis and methanogenesis during the anaerobic digestion of municipal solid waste (MSW) . The model was calibrated on previously published experimental data generated in 2-L reactors filled with shredded refuse and operated with leachate recirculation and neutralization . Based on model simulations, both waste degradation and methane production were stimulated when inhibition was prevented rapidly from the start, throughout the reactor volume, by leachate recirculation and neutralization . An optimal strategy to reduce the time needed for solid waste digestion is discussed . J Biomed Sci, 2002 Nov-Dec, 9(6 Pt 2), 631 - 8 Establishment of a human somatic hybrid cell line for recombinant protein production; Cho MS et al.; Cell fusion techniques were used to derive mammalian host cell lines suitable for large-scale production of therapeutic proteins . Although the 293S cell line, of human embryonic kidney origin, is an excellent host cell for mammalian gene expression, these cells have a tendency to form large and tight aggregates in suspension cultures and bioreactors . To solve the problem of aggregation, 293S cells were fused to a human suspension cell line, 2B8 (a Burkitt's lymphoma derivative), using polyethylene glycol (PEG) . The PEG-treated 293S and 2B8 cells were selected in a medium supplemented with hypoxanthine-aminopterin-thymidine and G418 (1 mg/ml) to eliminate nonfused cells . These hybrid clones, designated as HKB (hybrid of kidney and B cells), are negative for endogenous immunoglobulin expression . Most clones are readily adaptable to serum-free suspension culture under shaking conditions without forming large and tight aggregates . One clone, HKB11, was shown to support high-level expression of cytokines {interleukin (IL)-2 and IL-4}, ICAM-1 and rFVIII in a side-by-side comparison with 293 and Chinese hamster ovary cells . The above-described characteristics of HKB cells indicate that HKB11 is a favorable cell host for the production of human therapeutic proteins . Cells Tissues Organs, 2002, 172(2), 96 - 104 Skin genetically engineered as a bioreactor or a 'metabolic sink'; Christensen R et al.; Genetically manipulated human keratinocytes can produce and secrete medically relevant proteins to the circulation . Genetically modified skin may also function as a 'metabolic sink' detoxifying the body of metabolites which accumulate in certain metabolic diseases . At the National Institutes of Health (NIH), Bethesda, Md., a clinical trial investigating the treatment of an ocular disease using the skin as a 'metabolic sink' for ornithine accumulating in gyrate atrophy patients is being prepared . The trial will involve the transplantation of a small patch of autologous keratinocytes, transduced ex vivo, onto the thighs of patients with gyrate atrophy . We are now investigating other diseases where this technology may be applicable such as in the treatment of hyperphenylalaninemia or hypercholesterolemia . J Biotechnol, 2003 Jan 23, 100(2), 141 - 6 Progress towards a controlled culture of the marine sponge Pseudosuberites andrewsi in a bioreactor; Osinga R et al.; Explants of the tropical sponge Pseudosuberites andrewsi were fed with the marine diatom Phaeodactylum tricornotum . The food was supplied either as intact algae or as a filtered crude extract . Growth (measured as an increase in underwater weight) was found in both experiments . The explants fed with intact algae increased to an average underwater weight of 255% of the initial weight in 45-60 days . The explants fed with crude extract increased to an average of 200% of the initial weight in 30 days . These results show that it is possible to grow a sponge using a single microorganism species as a food source . In addition, it was demonstrated that sponges are also capable of growing on non-particulate food . Therefore, this study is an important step forward towards the development of controlled, in vivo sponge cultures. Folia Microbiol (Praha), 2002, 47(4), 417 - 21 Production of manganese-dependent peroxidase in a new solid-state bioreactor by Phanerochaete chrysosporium grown on wood shavings . Application to the decolorization of synthetic dyes; Rodriguez Couto S et al.; The production of manganese-dependent peroxidase (MnP) by Phanerochaete chrysosporium in a new solid-state bioreactor, the immersion bioreactor, operating with lignocellulosic waste, such as wood shavings, was investigated . Maximum MnP and lignin peroxidase (LiP) activity of 13.4 and 8.48 mukat/L were obtained, respectively . The in vitro decolorization of several synthetic dyes by the extracellular liquid produced in the above-mentioned bioreactor (containing mainly MnP) was carried out and its degrading ability was assessed . The highest decolorization was reached with Indigo Carmine (98%) followed by Bromophenol Blue (56%) and Methyl Orange (36%), whereas Gentian Violet was hardly decolorized (6%). Water Res, 2002 Nov, 36(18), 4605 - 15 Predicting oxygen transfer and water flow rate in airlift aerators; Burris VL et al.; Water flow rate, gas-phase holdup, and dissolved oxygen (DO) profiles are measured in a full-scale airlift aerator as a function of applied air flow rate . A model that predicts oxygen transfer based on discrete-bubble principles is applied . The riser DO profiles are used to calculate the initial bubble size . The range of calculated bubble diameters obtained using the model is 2.3-3.1 mm . The Sauter-mean diameter of bubbles measured in the laboratory ranged from 2.7 to 3.9 mm . The riser and downcomer DO profiles and gas holdups predicted by the model are in close agreement with the experimental results . A model that predicts water flow rate based on an energy balance is used to calculate Kt, the frictional loss coefficient for the air-water separator . Excluding the data at the very lowest air flow rate, the range of calculated values for Kt (3-8) is close to a literature value of 5.5 proposed for hydrodynamically similar external airlift bioreactors . The models should prove useful in the design and optimization of airlift aerators. Water Res, 2002 Nov, 36(18), 4445 - 54 Enzyme production activity of Phanerochaete chrysosporium and degradation of pentachlorophenol in a bioreactor; Shim SS et al.; Lignin peroxidase production by a white rot fungus, Phanerochaete chrysosporium, was experimentally investigated using a batch system and a reactor system with various carriers . Immobilization of mycelia cell culture was more effective in promoting cell growth and lignin peroxidase production compared to conventional stationary liquid culture . Biostage carrier, commonly used for biochemical treatment in a fluidized bed disposal system, greatly improved production of lignin peroxidase up to 8.1 U/mL in the batch system . The packed bed reactor system was operated using a repeated batch technique, consisting of alternating growth and production phases, to sustain lignin peroxidase growth and production during the entire experiment period . Steady-state continuous PCP degradation over an extended period was accomplished with a mineralization ratio exceeding 80% . These systems and operation methods are promising techniques for the treatment of hazardous waste. J Biotechnol, 2003 Jan 9, 100(1), 13 - 22 Plant-cell bioreactors with simultaneous electropermeabilization and electrophoresis; Yang RY et al.; Experimental investigations on using low-level electric currents and voltages to extract, transport, and collect intracellular secondary metabolites from plant cells while maintaining their viabilities were conducted focusing on the production of: (1) ionic betalains, mainly negatively-charged betanin, from Beta vulgaris cells, and (2) ionic alkaloids, particularly positively-charged ajmalicine and yohimbine, from Catharanthus roseus cells . Three versions of tubular membrane reactors in which electropermeabilization of cell membranes and electrophoresis and diffusion of ionic products take place simultaneously, with or without convective flow, to achieve desirable extraction were developed . Concentrations of secondary metabolites produced from these plant-cell reactors under steady and oscillatory electrical forcings were recorded and the viabilities of treated cells examined . Oscillatory application of electrical field appears to produce more products while retaining higher cell viability. J Ind Microbiol Biotechnol, 2002 Nov, 29(5), 268 - 74 Effect of nickel deprivation on methanol degradation in a methanogenic granular sludge bioreactor; Zandvoort MH et al.; The effect of omitting nickel from the influent on methanol conversion in an Upflow Anaerobic Sludge Bed (UASB) reactor was investigated . The UASB reactor (30 degrees C, pH 7) was operated for 261 days at a 12-h hydraulic retention time (HRT) and at organic loading rates (OLRs) ranging from 2.6 to 7.8 g COD l reactor(-1) day(-1) . The nickel content of the sludge decreased by 66% during the 261-day reactor run because of washout and doubling of the sludge bed volume . Nickel deprivation initially had a strong impact on the methanogenic activity of the sludge with methanol; e.g., after 89 days of operation, this activity was doubled by adding 2 micro M nickel . Upon prolonged UASB reactor operation, methanol and VFA effluent concentrations decreased whereas the sludge lost its response to nickel addition in activity tests . This suggests that a less nickel-dependent methanol-converting sludge had developed in the UASB reactor. J Ind Microbiol Biotechnol, 2002 Nov, 29(5), 259 - 63 Comparative studies on extracellular protease secretion and glucoamylase production by free and immobilized Aspergillus niger cultures; Papagianni M et al.; The effects of cell immobilization on the secretion of extracellular proteases and glucoamylase production by Aspergillus niger were investigated under a variety of immobilization techniques and culture conditions . Immobilization was achieved by means of cell attachment on metal surfaces or spore entrapment and subsequent growth on porous Celite beads . Free-suspension cultures were compared with immobilized mycelium under culture conditions that included growth in shake flasks and an airlift bioreactor . Cell attachment on metal surfaces minimized the secretion of proteases while enhancing glucoamylase production by the fungus . Growth on Celite beads in shake-flask cultures reduced the specific activity of the secreted proteases from 128 to 61 U g(-1), while glucoamylase specific activity increased from 205 to 350 U g(-1) . The effect was more pronounced in bioreactor cultures . A reduction of six orders of magnitude in protease specific activities was observed when the fungus grew immobilized on a rolled metal screen, which served as the draft tube of an airlift bioreactor. Appl Environ Microbiol, 2002 Nov, 68(11), 5367 - 73 RNA stable isotope probing, a novel means of linking microbial community function to phylogeny; Manefield M et al.; Identifying microorganisms responsible for recognized environmental processes remains a great challenge in contemporary microbial ecology . Only in the last few years have methodological innovations provided access to the relationship between the function of a microbial community and the phylogeny of the organisms accountable for it . In this study stable-isotope-labeled {13C}phenol was fed into a phenol-degrading community from an aerobic industrial bioreactor, and the 13C-labeled RNA produced was used to identify the bacteria responsible for the process . Stable-isotope-labeled RNA was analyzed by equilibrium density centrifugation in concert with reverse transcription-PCR and denaturing gradient gel electrophoresis . In contradiction with findings from conventional methodologies, this unique approach revealed that phenol degradation in the microbial community under investigation is dominated by a member of the Thauera genus . Our results suggest that this organism is important for the function of this bioreactor. Altern Lab Anim, 2002 Sep-Oct, 30(5), 525 - 38 Three-dimensional co-culture of primary human liver cells in bioreactors for in vitro drug studies: effects of the initial cell quality on the long-term maintenance of hepatocyte-specific functions; Zeilinger K et al.; In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry . A bioreactor culture model was developed that permits the three-dimensional co-culture of liver cells under continuous medium perfusion with decentralised mass exchange and integral oxygenation . We tested the ability of the system to support the long-term maintenance and differentiation of primary human liver cells . The effects of the initial cell quality were investigated by comparing cultures from resected, non-preserved liver with cultures from liver graft tissue damaged by long-term preservation . In cultures originating from non-preserved liver, protein and urea synthesis, glucose metabolism, and cytochrome (CYP450) activities were stable over the 2-week culture period, with maximal activities at the end of the first week in culture . Enzyme induction led to increased 7-ethoxyresorufin O-deethylase activities of up to 20 times the basal value . In cultures from preservation-damaged liver, recovery of metabolic activities was detected during bioreactor culture . After two weeks, most biochemical parameters approached those of cultures from non-preserved human liver . Light microscopy demonstrated the three-dimensional reorganisation of hepatocytes and non-parenchymal cells in co-culture . Long-term maintenance, and even the regeneration of specific functional activities of human liver cells, can be achieved in the bioreactor . This could facilitate the introduction into the pharmaceutical industry of in vitro drug testing with primary human liver cells. Altern Lab Anim, 2002 Sep-Oct, 30(5), 515 - 23 Long-term in vitro toxicity models: comparisons between a flow-cell bioreactor, a static-cell bioreactor and static cell cultures; Pazos P et al.; In vitro long-term toxicity testing is becoming an important issue in the field of toxicology, and there is a need to develop new model systems that mimic human chronic exposure and its effects . The aim of this work was to test two long-term in vitro toxicity systems which are available, a flow-cell bioreactor (Tecnomouse, Integra, Wallisellen, Switzerland) and a static cell bioreactor system (CELLine CL 6-well, Integra), and to compare them with the use of conventional cell culture flasks . A human cell line, Int 407, was exposed to cadmium chloride (CdCl(2); 10-(7-)10-(8)M) for 4 weeks . Cell numbers and cell viabilities were determined by the trypan blue (TB) exclusion assay and from exclusion of propidium iodide (PI) as determined by flow cytometry; and cell viability and metabolic activity were determined by the MTT assay . In addition, total protein determination and cadmium uptake measurements were performed . The results obtained with TB and PI exclusion did not show clear differences in cell viability with increasing CdCl(2) concentration . However, in the static cell-culture systems, an increase in MTT reduction was found at low concentrations of CdCl(2) . Expression of heat-shock protein (Hsp27 and Hsp70) increased differently, depending on the CdCl(2) concentration applied and the system used . In summary, of the two bioreactors, the CELLine CL 6-well bioreactor was shown to be the more efficient system for performing long-term cytotoxicity studies . It is easy to handle, it permits the assessment of several endpoints, and sufficient replicates can be made available. Biometals, 2002 Dec, 15(4), 377 - 90 Biotechnological potential of immobilized algae for wastewater N, P and metal removal: a review; Mallick N; This presentation comprises a review on the use of immobilized algae for wastewater nitrogen, phosphorus and metal removal purposes . Details of the use of immobilized algae, the techniques of immobilization and the effects of immobilization on cell function are included . Particularly relevant in their use for heavy metal removal from wastewaters; upon enriching the biomass in metal, can be recoverd, thereby providing economic advantages . The use of immobilized microalgae in these processes is very adequate and offers significant advantages in bioreactors . The future of this area of algal cell biotechnology is considered. Water Res, 2002 Sep, 36(16), 3941 - 8 A tanks-in-series bioreactor to simulate macromolecule-laden wastewater pretreatment under sewer conditions by Aspergillus niger; Coulibaly L et al.; Sewers are typically a means of transporting wastewater to a treatment facility, with little biotransformation of the soluble polymeric organic matter by suspended biomass . In the interest of providing an effective pretreatment of wastewater in a sewer network, it is necessary to design an accurate tool simulating sewer conditions and introduce an appropriate biomass for macromolecular pollutant degradation . Such a model reactor was built using a tanks-in-series design and the degradation of a polysaccharide (starch) by Aspergillus niger MUCL 28817 was studied . Starch degradation and the accumulation of intermediates (hydrolysis fragments) in the individual reactors were quantified under transient conditions, at a mean hydraulic residence time of 17 h . Starch was degraded by 90% in this reactor system and an accumulation of oligosaccharides with molecular weight lower than 1,000 Da was observed . These results may be helpful in the development of wastewater treatment in sewers and in the alleviation of the burden on undersized wastewater treatment systems. Biotechnol Bioeng, 2002 Dec 30, 80(7), 794 - 805 Modeling, optimization and experimental assessment of continuous L-(-)-carnitine production by Escherichia coli cultures; Alvarez-Vasquez F et al.; In a previous paper Canovas et al . (Biotechnol Bioeng 2002;77:764-775) presented a model for L-(-)-carnitine production using Escherichia coli O44 K74, in a cell-recycle bioreactor for the biotransformation of crotonobetaine into L-carnitine . In this work we optimize this biotechnological setup and experimentally verify the predicted optimal parameter profiles . Provided with a reliable and robust S-system description of the cell-bioreactor combined system, we applied the Indirect Optimization Method described by Torres et al . (Biotechnol Bioeng 1997;55(5):758-772; Food Technol Biotechnol 1998;36(3):177-184) . This optimization approach provides different parameter value profiles, all of which are compatible with the cell physiology and the bioreactor operating conditions, that yield increased rates of L-(-)-carnitine production . Three parameters were seen to be of critical importance for maximizing L-(-)-carnitine production: the dilution rate, the initial crotonobetaine concentration, and the carnitine dehydratase activity . When the first two were changed in the experimental setup, there was a 74% increase in the L-(-)-carnitine production rate, performance that was in close agreement with the predictions of the model . In accordance with the optimized solution, a further improvement (90% increase in the L-(-)-carnitine production rate) could be attained by over-expressing up to 5 times the carnitine dehydratase basal activity . Thus the optimization approach shown herein provides experimental evidence of a new strategy which demonstrates the possible variables that can be subjected to modifications compatible with the cell physiology and bioreactor operating conditions, and which are able to yield increased rates of L-(-)-carnitine production . Biotechnol Bioeng, 2002 Dec 30, 80(7), 746 - 54 Process control for enhanced L-phenylalanine production using different recombinant Escherichia coli strains; Gerigk M et al.; A novel fed-batch approach for the production of L-phenylalanine (L-Phe) with recombinant E . coli is presented concerning the on-line control of the key fermentation parameters glucose and tyrosine . Two different production strains possessing either the tyrosine feedback resistant aroF(fbr) (encoding tyrosine feedback resistant DAHP-synthase (3-desoxy-D-arabino-heptusonate-7-phosphate)) or the wild-type aroF(wt) were used as model systems to elucidate the necessity of finding an individual process optimum for each genotype . With the aid of tyrosine control, wild-type aroF(wt) could be used for L-Phe production achieving higher final L-Phe titers (34 g/L) than the aroF(fbr) strain (28 g/L) and providing higher DAHP-synthase activities . With on-line glucose control, an optimum glucose concentration of 5 g/L could be identified that allowed a sufficient carbon supply for L-Phe production while at the same time an overflow metabolism leading to acetate by-product formation was avoided . The process approach is suitable for other production strains not only in lab-scale but also in pilot-scale bioreactors . Biotechnol Bioeng, 2002 Dec 30, 80(7), 719 - 30 Analysis of the role of GADD153 in the control of apoptosis in NS0 myeloma cells; Lengwehasatit I et al.; Apoptosis can limit the maximum production of recombinant protein expression from cultured mammalian cells . This article focuses on the links between nutrient deprivation, ER perturbation, the regulation of (growth arrest and DNA damage inducible gene 153) GADD153 expression and apoptosis . During batch culture, decreases in glucose and glutamine correlated with an increase in apoptotic cells . This event was paralleled by a simultaneous increase in GADD153 expression . The expression of GADD153 in batch culture was suppressed by the addition of nutrients and with fed-batch culture the onset of apoptosis was delayed but not completely prevented . In defined stress conditions, glucose deprivation had the greatest effect on cell death when compared to glutamine deprivation or the addition of tunicamycin (an inhibitor of glycosylation), added to generate endoplasmic reticulum stress . However, the contribution of apoptosis to overall cell death (as judged by morphology) was smaller in conditions of glucose deprivation than in glutamine deprivation or tunicamycin treatment . Transient activation of GADD153 expression was found to occur in response to all stresses and occurred prior to detection of the onset of cell death . These results imply that GADD153 expression is either a trigger for apoptosis or offers a valid indicator of the likelihood of cell death arising from stresses of relevance to the bioreactor environment . Curr Opin Crit Care, 2002 Apr, 8(2), 171 - 7 Extracorporeal support of the failing liver; Mitzner SR et al.; Successes in machine-based extracorporeal support for different organ functions stimulated research in the field of liver support approximately 50 years ago . Initial failure to improve outcome using detoxification methods like dialysis, blood and plasma exchange, or plasmapheresis over sorbents fueled interest in biologic liver support concepts using bioreactors or combined methods . New device configurations, technical improvement of existing detoxification methods, and the refinement in cell culture techniques led to a boost in research on biologic and nonbiologic approaches . Currently, many systems are in the preclinical phase or have entered clinical studies . A number of completed clinical trials have reported a favorable therapeutic impact of the most advanced solutions on the course and outcome of liver failure . Often, findings must be reconfirmed . However, current knowledge suggests that extracorporeal liver support can successfully stabilize liver function, improve the clinical condition of patients, and considerably improve survival in certain subgroups of patients with fulminant hepatic failure and acute decompensation of chronic hepatic failure . Although the initial focus of liver support methods was bridging to liver transplantation, bridging to recovery of organ function and treatment of intractable pruritus are now valuable indications. J Biotechnol, 2002 Nov 13, 99(3), 249 - 57 Enzymatic membrane reactors for biodegradation of recalcitrant compounds . Application to dye decolourisation; Lopez C et al.; Membrane bioreactors are being increasingly used in enzymatic catalysed transformations . However, the application of enzymatic-based treatment systems in the environmental field is rather unusual . The aim of this paper is to overview the application of enzymatic membrane reactors to wastewater treatment, more specifically to dye decolourisation . Firstly, the basic aspects such as different configurations of enzymatic reactors, advantages and disadvantages associated to their utilisation are revised as well as the application of this technology to wastewater treatment . Secondly, dye decolourisation by white-rot fungi and their oxidative enzymes are discussed, presenting an overall view from for in vivo and in vitro systems . Finally, dye decolourisation by manganese peroxidase in an enzymatic membrane reactor in continuous operation is presented. J Biotechnol, 2002 Nov 13, 99(3), 237 - 48 Sensor fusion for on-line monitoring of yoghurt fermentation; Cimander C et al.; Measurement data from an electronic nose (EN), a near-infrared spectrometer (NIRS) and standard bioreactor probes were used to follow the course of lab-scale yoghurt fermentation . The sensor signals were fused using a cascade neural network: a primary network predicted quantitative process variables, including lactose, galactose and lactate; a secondary network predicted a qualitative process state variable describing critical process phases, such as the onset of coagulation or the harvest time . Although the accuracy of the neural network prediction was acceptable and comparable with the off-line reference assay, its stability and performance were significantly improved by correction of faulty data . The results demonstrate that on-line sensor fusion with the chosen analyzers improves monitoring and quality control of yoghurt fermentation with implications to other fermentation processes. Water Sci Technol, 2002, 46(6-7), 199 - 206 Non-point source pesticide removal by a mountainous wetland; Kao CM et al.; Non-point source (NPS) pollution is believed to be one of the major causes of impairment of water bodies . Among NPS pollution, agricultural NPS pollution is considered to be the largest single category resulting in water quality deterioration . Pesticides are some the most ubiquitous of these agricultural NPS pollutants . In this study, a mountainous wetland was selected to investigate the effects of the natural wetland system on the NPS pesticide (atrazine) removal to maintain the surface water quality . The selected wetland receives water from two unnamed creeks, which drain primarily upgradient agricultural lands . Wetland investigation and monitoring were conducted from November 1999 to March 2001 . Major storm events and baseline water quality samples were analyzed . Field results indicate that the wetland was able to remove NPS atrazine flushed from the upgradient agricultural lands after the occurrence of storm events . Laboratory aerobic and anaerobic bioreactor experiments were conducted to evaluate the biodegradation of atrazine under the intrinsic conditions of the wetland system . Microbial enumeration was conducted for a quick screen of bacterial activity in the studied wetland . Results from the study suggest that the methanogenesis process was possibly the dominant biodegradation pattern, and atrazine can be degraded under reductive dechlorinating conditions when sufficient intrinsic organic matter was provided . Results from this study can provide us with further knowledge on pesticide removal mechanisms in natural wetlands and evaluate the role of wetlands in controlling pesticide pollutants from stormwater runoff. Biotechnol Bioeng, 2002 Dec 20, 80(6), 677 - 84 Accurate and rapid viability assessment of Trichoderma harzianum using fluorescence-based digital image analysis; Hassan M et al.; Fluorescence microscopy and image analysis were evaluated in order to assess the viability of Trichoderma harzianum, an economically important filamentous fungus . After the evaluation of the two most commonly used fluorochromes, acridine orange (AO) and fluorescein diacetate (FDA) as metabolic indicator stains, AO gave ambiguous results and therefore FDA was chosen . The lower stability at room temperature and fast fluorescence intensity decay (50% after only 30 s of illumination in UV light) could be overcome by the use of a digital image acquisition system including frame grabber and a video camera . Fresh (live) fungal hyphae emitted bright green fluorescence when stained with this dye (7.5 microg/L), whereas a total absence of fluorescence was observed when using sterilized (dead) fungal cells . Fresh cells were subjected to different lethal and sublethal treatments and the percentage of FDA stained fluorescent hyphae was then measured over the total hyphal area (% of FDA-stained area) by image analysis . At the same time, samples were cultivated in shake flasks in order to correlate this % of FDA-stained area with its growth rate, a functional indicator of viability . The linear correlation (r = 0.979) was: growth rate (g/L x h) = 2.25 x 10(-3) (% of FDA-stained area) . This method was used to evaluate the viability of the fungus under two different fermentation conditions in a 10-L bioreactor . Estimated viable biomass during fermentation was strongly influenced by the process conditions . The use of FDA, with computer-aided quantitative image analysis, has made it possible to rapidly and reliably quantify the viability of T . harzianum . Biotechnol Bioeng, 2002 Dec 20, 80(6), 632 - 6 New bioreactor-coupled rapid stopped-flow sampling technique for measurements of metabolite dynamics on a subsecond time scale; Buziol S et al.; Knowledge of concentrations of intracellular metabolites is important for quantitative analysis of metabolic networks . As far as the very fast response of intracellular metabolites in the millisecond range is concerned, the frequently used pulse technique shows an inherent limitation . The time span between the disturbance and the first sample is constrained by the time necessary to obtain a homogeneous distribution of the pertubation within the bioreactor . For determination of rapid changes, a novel sampling technique based on the stopped-flow method has been developed . A continuous stream of biosuspension leaving the bioreactor is being mixed with a glucose solution in a turbulent mixing chamber . Through computer-aided activation of sequentially positioned three-way valves, different residence times and thus reaction times can be verified . The application of this new sampling method is illustrated with examples including measurements of adenine nucleotides and glucose-6-phosphate in Saccharomyces cerevisiae as well as measurements related to the PTS system in Escherichia coli . Huan Jing Ke Xue, 2002 Jul, 23(4), 67 - 70 {Studies on ceramic dual function membrane bioreactor for wastewater treatment}; Zhang Y et al.; A new bioreactor including ceramic dual function membrane, filtration and aeration, was developed and used for wastewater treatment . The turbidity of effluent by the ceramic membrane was comparable to that of 4-24 hours by sedimentation . Aeration and filtration could be switched constantly to the ceramic membrane in the bioreactor and the clogging of ceramic membrane could be well prevented . The wastewater was treated in batch and continues operation modes respectively and the experimental results showed that the COD loading was 1.1 kg/(m3.d) and 1.5 kg/(m3.d) respectively. Huan Jing Ke Xue, 2002 Jul, 23(4), 42 - 6 {Treatment of a 2,4-dichlorophenol contaminated wastewater in an air-lift inner-loop bioreactor}; Quan X et al.; An air lift inner-loop bioreactor packed with honeycomb-like ceramic carrier was immobilized with a 2,4-Dichlorophenol-degrading pure culture and was investigated to degrade 2,4-Dichlorophenol and phenol . In fed-batch operation mode, 2,4-DCP biodegradation rate increased with run numbers and followed zero-order kinetics model when it existed alone, but when 2,4-DCP was present in the mixture with phenol, phenol degradation rate had an apparent trend to increase whereas 2,4-DCP removal rate became slower and slower . In continuous operation, 2,4-DCP at the concentration ranged from 6.9 to 102.4 mg/L could be degraded well at the dilution rate of 0.16 h-1 and the average removal percentage was 96.5% . Carbon sources changed from 2,4-DCP to acetate sodium and peptone in the course of operation for 12 days did not cause the bacteria loss the DCP-degrading ability. Exp Dermatol, 2002 Oct, 11(5), 456 - 61 Keratinocyte gene therapy: cytokine gene expression in local keratinocytes and in circulation by introducing cytokine genes into skin; Meng X et al.; Using the plasmid DNA injection method, we introduced cytokine genes into skin to determine whether systemic expression of cytokine genes is possible . Eight human cytokine {interleukin-4 (IL-4), IL-6, IL-10, transforming growth factor beta1 (TGF-beta1), monocyte chemotactic and activating factor (MCAF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma)} gene expression vectors were constructed and injected into rat skin . Transgenic cytokines in local keratinocytes and in the sera were assayed with ELISA . Our results showed that transgenic cytokines were markedly increased in keratinocytes at the injection site . The serum concentrations of IL-4, 6, 10 and TauGF-beta1 reached levels high enough to have systemic biologic effects . However, other cytokines used in this study could not be detected in the sera . Moreover, the serum transgenic IL-10 level after subcutaneous injection was significantly higher than after intramuscular injection . We suggest that keratinocytes can be used as a bioreactor to achieve systemic expression of cytokine genes by DNA injection, but the transgenic protein level in circulation depends on different kinds of cytokine . This level also depends on different target cells used for gene transfer. J Gravit Physiol, 2001 Dec, 8(2), 57 - 66 Simulated conditions of microgravity suppress progesterone production by luteal cells of the pregnant rat; Bhat GK et al.; The purpose of this study was to assess whether simulated conditions of microgravity induce changes in the production of progesterone by luteal cells of the pregnant rat ovary using an in vitro model system . The microgravity environment was simulated using either a high aspect ratio vessel (HARV) bioreactor with free fall or a clinostat without free fall of cells . A mixed population of luteal cells isolated from the corpora lutea of day 8 pregnant rats was attached to cytodex microcarrier beads (cytodex 3) . These anchorage dependent cells were placed in equal numbers in the HARV or a spinner flask control vessel in culture conditions . It was found that HARV significantly reduced the daily production of progesterone from day 1 through day 8 compared to controls . Scanning electron microscopy showed that cells attached to the microcarrier beads throughout the duration of the experiment in both types of culture vessels . Cells cultured in chamber slide flasks and placed in a clinostat yielded similar results when compared to those in the HARV . Also, when they were stained by Oil Red-O for lipid droplets, the clinostat flasks showed a larger number of stained cells compared to control flasks at 48 h . Further, the relative amount of Oil Red-O staining per milligram of protein was found to be higher in the clinostat than in the control cells at 48 h . It is speculated that the increase in the level of lipid content in cells subjected to simulated conditions of microgravity may be due to a disruption in cholesterol transport and/or lesions in the steroidogenic pathway leading to a fall in the synthesis of progesterone . Additionally, the fall in progesterone in simulated conditions of microgravity could be due to apoptosis of luteal cells. Gene Ther, 2002 Oct, 9(20), 1351 - 8 Poly-L-glutamate, an anionic polymer, enhances transgene expression for plasmids delivered by intramuscular injection with in vivo electroporation; Nicol F et al.; Intramuscular (i.m.) injection of plasmids followed by electropermeabilization is an efficient process to deliver genes into skeletal myofibers that permits proteins to be produced and secreted at therapeutically relevant levels . To further improve skeletal muscle as a bioreactor, we identified a formulation that elevates transgene expression in myofibers after i.m . injection and electroporation . With secreted placental alkaline phosphate (SEAP) as reporter gene, plasmid formulated with poly-L-glutamate produced two- to eight-fold higher levels of SEAP in mouse serum than plasmid in saline . Various concentrations and molecular weights of poly-L-glutamate were similarly effective, but 6 mg/ml of 15-50 kDa poly-L-glutamate consistently yielded the highest expression levels . The poly-L-glutamate formulation was effective in two different muscle groups in mice at various plasmid doses for several transgenes, including an erythropoietin (EPO) gene, for which expression was elevated four- to 12-fold in comparison to animals that received EPO plasmid in saline . Transgene expression was localized to myofibers . Poly-L-glutamate may improve transgene expression in part by increasing plasmid retention in skeletal muscle . Poly-L-glutamate did not enhance gene transfer in the absence of electroporation . Therefore, the polymer is a novel formulation that specifically enhances the transfer and expression of genes delivered with electroporation. Biotechnol Prog, 2002 Sep-Oct, 18(5), 1003 - 9 Continuous selective extraction of secondary metabolites from Catharanthus roseus hairy roots with silicon oil in a two-liquid-phase bioreactor; Tikhomiroff C et al.; A two-liquid-phase bioreactor was designed to extract indole alkaloids from Catharanthus roseus hairy roots with silicon oil . Partition studies between silicon oil and culture medium showed that the silicon oil did not alter the availability of nutrients . The affinity of tabersonine and lochnericine for silicon oil is nine times higher than for the aqueous phase . Cultures were elicited with 25 mg/L of jasmonic acid . The growth of the hairy roots was not significantly modified by the presence of silicon oil . The overall specific yields of tabersonine and lochnericine were increased by 100-400% and 14-200%, respectively, with the use of silicon oil in nonelicited control cultures . In elicited cultures, these values were 10-55% for tabersonine and 20-65% for lochnericine . Serpentine was never found in the silicon oil . All measured alkaloids' specific yields were higher using silicon oil and elicitation, suggesting that the silicon oil, while acting as a metabolic sink for tabersonine and lochnericine, was efficient in increasing metabolic fluxes of the secondary metabolism pathways. Biotechnol Prog, 2002 Sep-Oct, 18(5), 975 - 85 Enzyme reaction engineering: effect of methanol on the synthesis of antibiotics catalyzed by immobilized penicillin G acylase under isothermal and non-isothermal conditions; Travascio P et al.; The effect of methanol on the kinetically controlled synthesis of cephalexin by free and immobilized penicillin G acylase (PGA) was investigated . Catalytic and hydrophobic membranes were obtained by chemical grafting, activation, and PGA immobilization on hydrophobic nylon supports . Butyl methacrylate (BMA) was used as graft monomer . Increasing concentrations of methanol were found to cause a greater deleterious effect on the activity of free than on that of the immobilized enzyme . Methanol, however, improved the kinetic stability of cephalexin synthesized by free PGA, resulting in higher maximum yields . By contrast, immobilized PGA reached 100% yields even in the absence of the cosolvent . Cephalexin synthesis by the catalytic membrane was also performed in a non-isothermal bioreactor . Under these conditions, a 94% increase of the synthetic activity and complete conversion of the limiting substrate to cephalexin were obtained . The addition of methanol reduced the non-isothermal activity increase . The physical cause responsible for the non-isothermal behavior of the hydrophobic catalytic membrane was identified in the process of thermodialysis. Biotechnol Prog, 2002 Sep-Oct, 18(5), 964 - 8 Polymerization of glucans by enzymatically active membranes; Becker M et al.; Conventional enzyme membrane reactors are not appropriate for a continuous synthesis of macromolecules and simultaneous product release . By immobilizing the enzyme in sufficiently large pores of a membrane an ensemble of miniaturized bioreactors is created . Product molecules are continuously removed from the enzyme by the flow of the reaction mixture across the membrane . Additionally, by varying the flow rate, it ought to be possible to influence the substrate as well as the enzyme-product residence times and thereby the product macromolecule's size . In this paper we present the first results of experiments involving enzymatic 1,4-alpha-glucan synthesis, using sucrose as substrate, maltooligosaccharides (DP 3-6) as primers, and membrane-immobilized amylosucrase . Epoxy groups for a covalent enzyme immobilization were generated on polypropylene microfiltration membranes by heterogeneous photoinitiated graft polymerization of glycidyl methacrylate . The influence of primer concentration and flow rate through the enzyme-membrane on amylosucrase activity, molecule growth, and coupling efficiency for glucose (% of coupled glucose versus free glucose) were investigated . The enzymatically mediated chain elongation of maltooligosaccharides by the successive addition of glucose units was achieved for the first time in a transmembrane process utilizing amylosucrase membranes. Biotechnol Prog, 2002 Sep-Oct, 18(5), 951 - 63 Computational fluid dynamics modeling of steady-state momentum and mass transport in a bioreactor for cartilage tissue engineering; Williams KA et al.; Computational fluid dynamics (CFD) models to quantify momentum and mass transport under conditions of tissue growth will aid bioreactor design for development of tissue-engineered cartilage constructs . Fluent CFD models are used to calculate flow fields, shear stresses, and oxygen profiles around nonporous constructs simulating cartilage development in our concentric cylinder bioreactor . The shear stress distribution ranges from 1.5 to 12 dyn/cm(2) across the construct surfaces exposed to fluid flow and varies little with the relative number or placement of constructs in the bioreactor . Approximately 80% of the construct surface exposed to flow experiences shear stresses between 1.5 and 4 dyn/cm(2), validating the assumption that the concentric cylinder bioreactor provides a relatively homogeneous hydrodynamic environment for construct growth . Species mass transport modeling for oxygen demonstrates that fluid-phase oxygen transport to constructs is uniform . Some O(2) depletion near the down stream edge of constructs is noted with minimum pO(2) values near the constructs of 35 mmHg (23% O(2) saturation) . These values are above oxygen concentrations in cartilage in vivo, suggesting that bioreactor oxygen concentrations likely do not affect chondrocyte growth . Scale-up studies demonstrate the utility and flexibility of CFD models to design and characterize bioreactors for growth of tissue-engineered cartilage. Acta Biochim Pol, 2002, 49(2), 313 - 21 Antiangiogenic gene therapy in inhibition of metastasis; Szala S et al.; This short review attempts to demonstrate the usefulness of antiangiogenic gene therapy in achieving inhibition of growth in experimentally-induced metastases . Certain normal tissues (for example skeletal muscle) may be used in vivo, after genetic modification, as a "bioreactor", able to produce and secrete into the bloodstream proteins known to exert antiangiogenic effects . By inhibiting neoangiogenesis these proteins would thus prevent the development of metastases . The review discusses also the perspectives of antimetastatic therapy based on certain types of allogenic cells (for example myoblasts and fibroblasts) that had been genetically modified and then microencapsulated . The strategy of encapsulation is aimed at protecting the modified cells secreting antiangiogenic factors from being eliminated by the immune system . Secretion of antiangiogenic proteins by these microencapsulated cells can be controlled with inducible promoters . Antiangiogenic genes remaining under the transcriptional control of such promoters may be switched on and off using antibiotics, such as tetracycline derivatives, or steroid hormones. Nutrition, 2002 Oct, 18(10), 842 - 8 Insulin secretion and sensitivity in space flight: diabetogenic effects; Tobin BW et al.; Nearly three decades of space flight research have suggested that there are subclinical diabetogenic changes that occur in microgravity . Alterations in insulin secretion, insulin sensitivity, glucose tolerance, and metabolism of protein and amino acids support the hypothesis that insulin plays an essential role in the maintenance of muscle mass in extended-duration space flight . Experiments in flight and after flight and ground-based bedrest studies have associated microgravity and its experimental paradigms with manifestations similar to those of diabetes, physical inactivity, and aging . We propose that these manifestations are characterized best by an etiology that falls into the clinical category of "other" causes of diabetes, including, but not restricted to, genetic beta-cell defects, insulin action defects, diseases of the endocrine pancreas, endocrinopathies, drug or chemically induced diabetes, infections, immune-mediated metabolic alteration, and a host of genetic related diseases . We present data showing alterations in tumor necrosis factor-alpha production, insulin secretion, and amino acid metabolism in pancreatic islets of Langerhans cultured in a ground-based cell culture bioreactor that mimics some of the effects of microgravity . Taken together, space flight research, ground-based studies, and bioreactor studies of pancreatic islets of Langerhans support the hypothesis that the pancreas is unable to overcome peripheral insulin resistance and amino acid dysregulation during space flight . We propose that measures of insulin secretion and insulin action will be necessary to design effective countermeasures against muscle loss, and we advance the "disposition index" as an essential model to be used in the clinical management of space flight-induced muscle loss. Biomaterials, 2002 Dec, 23(24), 4793 - 801 Cardiac tissue engineering: characteristics of in unison contracting two- and three-dimensional neonatal rat ventricle cell (co)-cultures; van Luyn MJ et al.; Patients with heart failure have, in spite of improved palliative therapies, bad prognosis . Cardiac tissue engineering by use of a temporary bioscaffold and cardiomyocytes may help to find answers for future treatments in heart failure . For that purpose two neonatal rat heart ventricular cell fractions were obtained after a gradient cell separation . Time related characteristics of Fractions I and II were established in two-dimensional (2-D) and three-dimensional (3-D) cell cultures . The 3-D cardiac constructs were obtained by use of a bovine type I collagen matrix after culturing either under static conditions or in the HARV bioreactor . With the 2-D cultures contracting cells were present after 1 day, and reached confluency from day 5 on and this was maintained up to 135 days . In Fraction-I some non-contracting cells were always noticed between the (in time in unison) contracting cells . Transmission electron microscopy (TEM) revealed that these mainly concerned fibroblasts . Differences in the expression of alpha-SM-1 actin and troponin-T were observed between the two fractions . In both fractions endothelial cells and macrophages were only sporadically observed . All through the 3-D matrix pendant-like single cell and clustered cell contractions were present after 1-2 days, resulting in time in unison contracting of cells with the collagen matrices . The whole event was faster with Fraction-I and was observed up to 3 weeks . At this time point clusters of troponin-T positive cells were found scattered through the collagen matrices . Additionally, TEM revealed healthy layers of connected cardiomyocytes with intercalated discs, in this case on and in between the collagen fibres . These findings provide evidence that in unison contracting structurally organized cell-matrix cardiac constructs can be engineered by use of co-cultures (neonatal cardiomyocytes and fibroblasts) and collagen matrices, which is very promising for the repair of larger scar areas of the myocardium . Water Sci Technol, 2002, 46(4-5), 67 - 76 Pilot-scale testing membrane bioreactor for wastewater reclamation in industrial laundry; Andersen M et al.; A pilot-scale study of membrane bioreactor treatment for reclamation of wastewater from Berendsen Textile Service industrial laundry in Soborg, Denmark was carried out over a 4 month period . A satisfactory COD degradation was performed resulting in a low COD in the permeate (< 50 mg/l) . To obtain satisfactory treatment, addition of nitrogen was necessary . The biodegradability of the permeate was very low (BOD5 < 2 mg/l) . A hydraulic retention time of 1 d turned out to be sufficient at a sludge concentration of 10 g MLSS/l . Through addition of a cationic polymer, a satisfactory dewaterability of the sludge was reached . Membrane tests showed that operating at a trans-membrane pressure of 3 bar and a cross-flow velocity of 4 m/s, a flux of 120 l/m2h can be expected without using chemicals for membrane cleaning . The quality of the permeate was very good when comparing to the reuse quality demands of water to the wash processes . Reuse of the permeate in all rinsing steps requires additional treatment through reverse osmosis. Water Sci Technol, 2002, 46(4-5), 281 - 6 Hanced biological phosphorus removal in membrane bioreactors; Adam C et al.; Enhanced biological phosphorus removal (Bio-P) in a membrane bioreactor (MBR) promises several advantages but was never attempted as not compatible with high sludge ages . This article includes description and results of bench-scale investigations on Bio-P removal in an MBR . An MBR bench-scale plant (210 L) was operated in parallel to a conventional WWTP under comparable process conditions . The results show that Bio-P removal is possible in MBR . The effluent qualities of the plants were comparable . The effluent P-concentration was always lower than 0.2 mg P(T)/L . In the MBR bench-scale plant P-uptake occurred mainly in the anoxic zone . Investigations with P-spiking showed higher Bio-P potential as P-removal increased up to 20-25 mg/L while P/TS rose up to >6%. Water Sci Technol, 2002, 46(4-5), 19 - 26 Coarse media filtration for enhanced primary treatment of municipal wastewater; Liao Z et al.; In these experimental studies enhanced primary treatment in coarse media filters has been evaluated . The coarse media used in the filters have been plastic carriers normally used for moving bed bioreactors, a Kaldnes carrier K1 with density < 1 g/cm3 (K1L) and a bigger carrier K2, that was used both in a lighter, floating (K2L) version and a heavier, sinking one (K2H) . The results demonstrated that media characteristics significantly influenced filter performance . The dosing of a high MW and high charge cationic polymer was, however, found to have an even greater effect on treatment efficiency and especially on head loss development . Both filters were found to be feasible for enhanced primary treatment at high filtration rates (20-30 m/h) especially when dosing low dosages (1-3 mg/l) of polymer . The combination of two media into a dual media filter might take the advantages of both . Further experiments were carried out on a dual media filter consisting of lighter K1L media and heavier K2H media . It was indeed found that the K1L+K2H up-flow filter resulted in better performances in terms of water production when an optimised backwash procedure was taken into account. Biotechnol Bioeng, 2002 Dec 5, 80(5), 569 - 79 Serum-free suspension cultivation of PER.C6(R) cells and recombinant adenovirus production under different pH conditions; Xie L et al.; PER.C6(R) cell growth, metabolism, and adenovirus production were studied in head-to-head comparisons in stirred bioreactors under different pH conditions . Cell growth rate was found to be similar in the pH range of 7.1-7.6, while a long lag phase and a slower growth rate were observed at pH 6.8 . The specific consumption rates of glucose and glutamine decreased rapidly over time during batch cell growth, as did the specific lactate and ammonium production rates . Cell metabolism in both infected and uninfected cultures was very sensitive to culture pH, resulting in dramatic differences in glucose/glutamine consumption and lactate/ammonium production under different pH conditions . It appeared that glucose metabolism was suppressed at low pH but the efficiency of energy production from glucose was enhanced . Adenovirus infection resulted in profound changes in cell growth and metabolism . Cell growth was largely arrested under all pH conditions, while glucose consumption and lactate production were elevated post virus infection . Virus infection induced a reduction in glutamine consumption at low pH but an increase at high pH . The optimal pH for adenovirus production was found to be 7.3 under the experimental conditions used in the study . Deviations from this optimum resulted in significant reductions of virus productivity . The results indicate that culture pH is a very critical process parameter in PER.C6(R) cell culture and adenovirus production . Circulation, 2002 Sep 24, 106(12 Suppl 1), I143 - 50 Tissue engineering of functional trileaflet heart valves from human marrow stromal cells; Hoerstrup SP et al.; BACKGROUND: We previously demonstrated the successful tissue engineering and implantation of functioning autologous heart valves based on vascular-derived cells . Human marrow stromal cells (MSC) exhibit the potential to differentiate into multiple cell-lineages and can be easily obtained clinically . The feasibility of creating tissue engineered heart valves (TEHV) from MSC as an alternative cell source, and the impact of a biomimetic in vitro environment on tissue differentiation was investigated . METHODS AND RESULTS: Human MSC were isolated, expanded in culture, and characterized by flow-cytometry and immunohistochemistry . Trileaflet heart valves fabricated from rapidly bioabsorbable polymers were seeded with MSC and grown in vitro in a pulsatile-flow-bioreactor . Morphological characterization included histology and electron microscopy (EM) . Extracellular matrix (ECM)-formation was analyzed by immunohistochemistry, ECM protein content (collagen, glycosaminoglycan) and cell proliferation (DNA) were biochemically quantified . Biomechanical evaluation was performed using Instron(TM) . In all valves synchronous opening and closing was observed in the bioreactor . Flow-cytometry of MSC pre-seeding was positive for ASMA, vimentin, negative for CD 31, LDL, CD 14 . Histology of the TEHV-leaflets demonstrated viable tissue and ECM formation . EM demonstrated cell elements typical of viable, secretionally active myofibroblasts (actin/myosin filaments, collagen fibrils, elastin) and confluent, homogenous tissue surfaces . Collagen types I, III, ASMA, and vimentin were detected in the TEHV-leaflets . Mechanical properties of the TEHV-leaflets were comparable to native tissue . CONCLUSION: Generation of functional TEHV from human MSC was feasible utilizing a biomimetic in vitro environment . The neo-tissue showed morphological features and mechanical properties of human native-heart-valve tissue . The human MSC demonstrated characteristics of myofibroblast differentiation. J Urol, 2002 Oct, 168(4 Pt 2), 1794 - 7 Engineering of human cartilage rods: potential application for penile prostheses; Kim BS et al.; PURPOSE: Natural penile prostheses created from the patient's own cells may eliminate the biocompatibility risks associated with artificial prostheses . We previously demonstrated that autologous cartilaginous rods could be created in animal corpus cavernosum as penile prostheses in situ by transplanting autologous chondrocytes on biodegradable polymer scaffolds . In the present study we investigated the possibility of engineering human cartilage rods for potential use as penile prostheses . MATERIALS AND METHODS: Chondrocytes isolated from human ear were seeded on rod shaped biodegradable polymer scaffolds (1.2 cm . in diameter, 6.0 cm . long) . The seeded scaffolds were maintained in stirred bioreactors for 1 month . Subsequently, the seeded scaffolds were implanted subcutaneously into athymic rats . The specimens were retrieved 2 months after implantation, and histological, structural and mechanical properties were analyzed . The mechanical properties of the engineered prostheses were compared to those of silicone prostheses . RESULTS: Human chondrocytes seeded onto polymer scaffolds formed milk-white cartilaginous rods of the same size as the initial implants . Histological analyses using hematoxylin and eosin, toluidine blue and alcian blue showed mature and well-formed chrondrocytes in the retrieved implants . The engineered human cartilaginous rods were flexible, elastic and able to withstand high degrees of compressive forces . The mechanical properties were comparable to those of commercially available silicone prostheses . CONCLUSIONS: Transplantation of chrondrocytes isolated from human ear on polymer scaffolds resulted in the formation of human cartilage rods with the appropriate mechanical properties required for use as penile prostheses . This study demonstrates the feasibility of creating human cartilage rods with a large dimension . This technology may be useful for patients who need penile reconstruction. Biotechnol Bioeng, 2002 Nov 20, 80(4), 465 - 76 RWPV bioreactor mass transport: earth-based and in microgravity; Begley CM et al.; Mass transport and mixing of perfused scalar quantities in the NASA Rotating Wall Perfused Vessel bioreactor are studied using numerical models of the flow field and scalar concentration field . Operating conditions typical of both microgravity and ground-based cell cultures are studied to determine the expected vessel performance for both flight and ground-based control experiments . Results are presented for the transport of oxygen with cell densities and consumption rates typical of colon cancer cells cultured in the RWPV . The transport and mixing characteristics are first investigated with a step change in the perfusion inlet concentration by computing the time histories of the time to exceed 10% inlet concentration . The effects of a uniform cell utilization rate are then investigated with time histories of the outlet concentration, volume average concentration, and volume fraction starved . It is found that the operating conditions used in microgravity produce results that are quite different then those for ground-based conditions . Mixing times for microgravity conditions are significantly shorter than those for ground-based operation . Increasing the differential rotation rates (microgravity) increases the mixing and transport, while increasing the mean rotation rate (ground-based) suppresses both . Increasing perfusion rates enhances mass transport for both microgravity and ground-based cases, however, for the present range of operating conditions, above 5-10 cc/min there are diminishing returns as much of the inlet fluid is transported directly to the perfusion exit . The results show that exit concentration is not a good indicator of the concentration distributions in the vessel . In microgravity conditions, the NASA RWPV bioreactor with the viscous pump has been shown to provide an environment that is well mixed . Even when operated near the theoretical minimum perfusion rates, only a small fraction of the volume provides less than the required oxygen levels . 2002 Wiley Periodicals, Inc. Biotechnol Bioeng, 2002 Nov 20, 80(4), 438 - 44 Scale-up and optimization of an acoustic filter for 200 L/day perfusion of a CHO cell culture; Gorenflo VM et al.; Acoustic cell retention devices have provided a practical alternative for up to 50 L/day perfusion cultures but further scale-up has been limited . A novel temperature-controlled and larger-scale acoustic separator was evaluated at up to 400 L/day for a 10(7) CHO cell/mL perfusion culture using a 100-L bioreactor that produced up to 34 g/day recombinant protein . The increased active volume of this scaled-up separator was divided into four parallel compartments for improved fluid dynamics . Operational settings of the acoustic separator were optimized and the limits of robust operations explored . The performance was not influenced over wide ranges of duty cycle stop and run times . The maximum performance of 96% separation efficiency at 200 L/day was obtained by setting the separator temperature to 35.1 degrees C, the recirculation rate to three times the harvest rate, and the power to 90 W . While there was no detectable effect on culture viability, viable cells were selectively retained, especially at 50 L/day, where there was a 5-fold higher nonviable washout efficiency . Overall, the new temperature-controlled and scaled-up separator design performed reliably in a way similar to smaller-scale acoustic separators . These results provide strong support for the feasibility of much greater scale-up of acoustic separations . Biotechnol Bioeng, 2002 Nov 20, 80(4), 428 - 37 Fabrication and use of a transient contractional flow device to quantify the sensitivity of mammalian and insect cells to hydrodynamic forces; Ma N et al.; A microfluidic device was fabricated via photolithographic techniques which can create transient elongational and shear forces ranging over three orders of magnitude while still maintaining laminar flow conditions . The contractional fluid flow inside the microfluidic device was simulated with FLUENT (a computational fluid dynamics computer program) and the local deformation forces were characterized with the scalar quantity, local energy dissipation rate . The sensitivities of four cell lines (CHO, HB-24, Sf-9, and MCF7) were tested in the device . The results indicate that all four cell lines are able to withstand relatively intense energy dissipation rates (up to 10(4)-10(5) kW/m(3)), which is orders of magnitude higher than the maximum local energy dissipation rates generated by impellers in bioreactors, but comparable to that associated with small bursting bubbles . While the concept that suspended animal cells are relatively robust with respect to purely hydrodynamic forces in bioprocess equipment is well known, these results quantitatively demonstrate these observations . Biotechnol Bioeng, 2002 Nov 20, 80(4), 405 - 13 Use of at-line and in-situ near-infrared spectroscopy to monitor biomass in an industrial fed-batch Escherichia coli process; Arnold SA et al.; One of the key goals in bioprocess monitoring is to achieve real-time knowledge of conditions within the bioreactor, i.e., in-situ . Near-infrared spectroscopy (NIRS), with its ability to carry out multi-analyte quantification rapidly with little sample presentation, is potentially applicable in this role . In the present study, the application of NIRS to a complex, fed-batch industrial E . coli (RV308/PHKY531) process was investigated . This process undergoes a series of temperature changes and is vigorously agitated and aerated . These conditions can pose added challenges to in-situ NIRS . Using the measurement of a key analyte (biomass) as an illustration, the details of the relationship between the at-line and in-situ use of NIRS are considered from the viewpoint of both theory and practical application . This study shows that NIRS can be used both at-line and in-situ in order to achieve good predictive models for biomass . There are particular challenges imposed by in-situ operation (loss of wavelength regions and noise) which meant the need for signal optimisation studies . This showed that whilst the at-line modelling process may provide some useful information for the in-situ process, there were distinct differences . This study shows that the in-situ use of NIRS in a highly challenging matrix (similar to those encountered in current industrial practice) is possible, and thus extends previous works in the area . J Virol Methods, 2002 Sep, 105(2), 321 - 30 Quantitation of baculovirus particles by flow cytometry; Shen CF et al.; A method using flow cytometry (FCM) analysis was developed to quantitate baculovirus total particles produced in insect cell cultures . The method is a direct count of particles and involves staining of the baculovirus DNA with SYBR Green I, a highly fluorescent nucleic acid specific dye . Sample preparation of cell-free supernatant containing budded viral particles involves fixation with paraformaldehyde, freeze-thaw treatment, viral membrane permeabilization with Triton X-100, and sample heating to improve staining efficiency and enhance baculovirus particle green fluorescence intensities . In this study, the effects of the different treatment steps and medium composition on viral particle counts were examined in order to identify optimal preparation conditions . FCM analysis linearity was established over a viral concentration range of two logs with a lower detection limit at 10(5) viral particles per ml . Robustness and reproducibility of the method were assessed using samples from large-scale bioreactor cultures . The events (or virus particle counts) obtained by FCM analysis were usually higher than the titres obtained by end-point dilution assay (EPDA) . Results from 16 different viral stocks showed an average ratio of 3.7 total particles (FCM) to infectious particles (EPDA) . Essentially, the FCM analysis reported below shortens baculovirus quantitation time to 2 h and provides a good estimation of virus titers . It is believed that these findings will contribute to acceleration of process development in the area of baculovirus expression technology in general and specifically in process where stoichiometric multi-viral infections of cells are critical to the expression of complex products. J Ind Microbiol Biotechnol, 2002 Sep, 29(3), 140 - 4 Continuous ethanol production in a nonconventional five-stage system operating with yeast cell recycling at elevated temperatures; Laluce C et al.; Three ranges of increasing temperatures (35-43, 37-45, 39-47 degrees C) were sequentially applied to a five-stage system continuously operated with cell recycling so that differences of 2 degrees C (between one reactor to the next) and 8 degrees C (between the first reactor at the highest temperature and the fifth at the lowest temperature) were kept among the reactors for each temperature range . The entire system was fed through the first reactor . The lowest values of biomass and viability were obtained for reactor R(3) located in the middle of the system . The highest yield of biomass was obtained in the effluent when the system was operated at 35-43 degrees C . This nonconventional system was set up to simulate the local fluctuations in temperature and nutrient concentrations that occur in different regions of the medium in an industrial bioreactor for fuel ethanol production mainly in tropical climates . Minimized cell death and continuous sugar utilization were observed at temperatures normally considered too high for Saccharomyces cerevisiae fermentations. Biotechnol Appl Biochem, 2002 Oct, 36(Pt 2), 127 - 31 Application of statistically based experimental designs for the optimization of exo-polysaccharide production by Cordyceps militaris NG3; Xu CP et al.; Statistically based experimental designs were applied to the optimization of medium composition for exo-polysaccharide production by Cordyceps militaris NG3 in shake-flask cultures . First, the Plackett-Burman design was used to search for the main factors on mycelia and exo-polysaccharide production . Among these variables, sucrose and corn steep powder were found to be two significant factors and had positive effects on mycelial yield (with confidence level >80%) and exo-polysaccharide production (with confidence level >90%) . Subsequently, to study the mutual interactions between variables, the effects of the two main factors on exo-polysaccharide production were further investigated using a central composite design . The optimal composition was found to be 1.03 g/l corn steep powder, 2.95 g/l sucrose, 0.1 g/l K(2)HPO(4), 0.5 g/l MgSO(4) x 5H(2)O and 0.1 g/l KNO(3) for the enhanced production of the exo-polysaccharide, which was 2.604 g/l in shake-flask cultures . Under optimal culture conditions, the maximum exo-polysaccharide concentration in a 5 l stirred-tank bioreactor was 3.8 g/l. Biotechnol Appl Biochem, 2002 Oct, 36(Pt 2), 95 - 100 Chlorpromazine oxidation by hydroperoxidase activity of covalent immobilized lipoxygenase; Santano E et al.; The application of the hydroperoxidase activity of immobilized lipoxygenase to xenobiotic metabolization is reported in this work . Soya bean lipoxygenase has been immobilized by covalent coupling to oxirane acrylic beads . This immobilized system has been applied to the oxidation of chlorpromazine (CPZ), a phenothiazine of wide pharmacological use which in some cases presents toxicity . Immobilized lipoxygenase produces the oxidation of CPZ in the presence of hydrogen peroxide at acidic pH, maintaining a high level of activity and stability . In comparison with free lipoxygenase, the immobilized enzyme system shows higher catalytic efficiency and protection against enzymic inactivation produced by the presence of H(2)O(2) . When the system of immobilized enzyme was loaded into a bioreactor operating in continuous mode, the level of CPZ oxidation was higher than obtained using a discontinuous procedure or the free enzyme . The results obtained in this work suggest that a system of covalent immobilized lipoxygenase operating in continuous mode may constitute a valuable tool for xenobiotic detoxification or metabolization studies. J Chromatogr A, 2002 Aug 30, 968(1-2), 113 - 20 On-line purification of His-tag enhanced green fluorescent protein taken directly from a bioreactor by continuous ultrasonic homogenization coupled with immobilized metal affinity expanded bed adsorption; Noubhani AM et al.; In this report, we describe a new process for the on-line purification of His-tag EGFP (enhanced green fluorescent protein) taken directly from a bioreactor by continuous ultrasonic homogenization coupled with immobilized metal affinity expanded bed adsorption (IMAEBA) . The use of proteins including a histidine-tag facilitates their subsequent purification after expression in many microorganisms . This meets the needs of scientific researchers as well as industrialists interested in purifying recombinant proteins . After evaluating the different flow-rates and ultrasonic probe sizes, the on-line purification was tested . After ultrasonic treatment, 70% of the cells were broken and 90% of free EGFP was recovered after IMAEBA . In our conditions, more than 450 mg of EGFP were obtained in 15 h . On-line bioreactor-ultrasonic probe-immobilized metal affinity expanded bed adsorption is a rapid automated technique for obtaining large quantities of pure EGFP. FASEB J, 2002 Oct, 16(12), 1691 - 4 Epub 2002 Aug 07. Bioreactors mediate the effectiveness of tissue engineering scaffolds; Pei M et al.; We hypothesized that the mechanically active environment present in rotating bioreactors mediates the effectiveness of three-dimensional (3D) scaffolds for cartilage tissue engineering . Cartilaginous constructs were engineered by using bovine calf chondrocytes in conjunction with two scaffold materials (SM) (benzylated hyaluronan and polyglycolic acid); three scaffold structures (SS) (sponge, non-woven mesh, and composite woven/non-woven mesh); and two culture systems (CS) (a bioreactor system and petri dishes) . Construct size, composition {cells, glycosaminoglycans (GAG), total collagen, and type-specific collagen mRNA expression and protein levels}, and mechanical function (compressive modulus) were assessed, and individual and interactive effects of model system parameters (SM, SS, CS, SM*CS and SS*CS) were demonstrated . The CS affected cell seeding (higher yields of more spatially uniform cells were obtained in bioreactor-grown than dish-grown 3-day constructs) and subsequently affected chondrogenesis (higher cell numbers, wet weights, wet weight GAG fractions, and collagen type II levels were obtained in bioreactor-grown than dish-grown 1-month constructs) . In bioreactors, mesh-based scaffolds yielded 1-month constructs with lower type I collagen levels and four-fold higher compressive moduli than corresponding sponge-based scaffolds . The data imply that interactions between bioreactors and 3D tissue engineering scaffolds can be utilized to improve the structure, function, and molecular properties of in vitro-generated cartilage. Hum Gene Ther, 2002 Sep 1, 13(13), 1621 - 30 Gene transfer of a human insulin-like growth factor I cDNA enhances tissue engineering of cartilage; Madry H et al.; The repair of articular cartilage lesions remains a clinical problem . Two novel approaches to cartilage formation, gene transfer and tissue engineering, have been limited by short-term transgene expression in transplanted chondrocytes and inability to deliver regulatory signals to engineered tissues according to specific temporal and spatial patterns . We tested the hypothesis that the transfer of a cDNA encoding the human insulin-like growth factor I (IGF-I) can provide sustained gene expression in cell-polymer constructs in vitro and in vivo and enhance the structural and functional properties of tissue-engineered cartilage . Bovine articular chondrocytes genetically modified to overexpress human IGF-I were seeded into polymer scaffolds, cultured in bioreactors in serum-free medium, and implanted subcutaneously in nude mice; constructs based on nontransfected or lacZ-transfected chondrocytes served as controls . Transgene expression was maintained throughout the duration of the study, more than 4 weeks in vitro followed by an additional 10 days either in vitro or in vivo . Chondrogenesis progressed toward the formation of cartilaginous tissue that was characterized by the presence of glycosaminoglycans, aggrecan, and type II collagen, and the absence of type I collagen . IGF-I constructs contained increased amounts of glycosaminoglycans and collagen and confined-compression equilibrium moduli as compared with controls; all groups had subnormal cellularity . The amounts of glycosaminoglycans and collagen per unit DNA in IGF-I constructs were markedly higher than in constructs cultured in serum-supplemented medium or native cartilage . This enhancement of chondrogenesis by spatially defined overexpression of human IGF-I suggests that cartilage tissue engineering based on genetically modified chondrocytes may be advantageous as compared with either gene transfer or tissue engineering alone. Biotechnol Bioeng, 2002 Nov 5, 80(3), 349 - 57 Effect of particle-particle shearing on the bioleaching of sulfide minerals; Chong N et al.; The biological leaching of sulfide minerals, used for the production of gold, copper, zinc, cobalt, and other metals, is very often carried out in slurry bioreactors, where the shearing between sulfide particles is intensive . In order to be able to improve the efficiency of the bioleaching, it is of significant importance to know the effect of particle shearing on the rate of leaching . The recently proposed concept of ore immobilization allowed us to study the effect of particle shearing on the rate of sulfide (pyrite) leaching by Thiobacillus ferrooxidans . Using this concept, we designed two very similar bioreactors, the main difference between which was the presence and absence of particle-particle shearing . It was shown that when the oxygen mass transfer was not the rate-limiting step, the rate of bioleaching in the frictionless bioreactor was 2.5 times higher than that in a bioreactor with particle friction (shearing) . The concentration of free suspended cells in the frictionless bioreactor was by orders of magnitude lower than that in the frictional bioreactor, which showed that particle friction strongly reduces the microbial attachment to sulfide surface, which, in turn, reduces the rate of bioleaching . Surprisingly, it was found that formation of a layer of insoluble iron salts on the surface of sulfide particles is much slower under shearless conditions than in the presence of particle-particle shearing . This was explained by the effect of particle friction on liquid-solid mass transfer rate . The results of this study show that reduction of the particle friction during bioleaching of sulfide minerals can bring important advantages not only by increasing significantly the bioleaching rate, but also by increasing the rate of gas-liquid oxygen mass transfer, reducing the formation of iron precipitates and reducing the energy consumption . One of the efficient methods for reduction of particle friction is ore immobilization in a porous matrix . Appl Microbiol Biotechnol, 2002 Sep, 59(6), 685 - 94 Epub 2002 Jul 17. Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors; Miksch G et al.; The gene for phytase from Escherichia coli was sequenced and compared with the appA gene . It was found to be a mutant derivative of the appA gene . After fusion with a C-terminal His-tag, phytase was purified by affinity chromatography and the enzymatic properties were analyzed . To develop a system for overexpression and extracellular production of phytase in E . coli, factors affecting the expression and secretion such as promoter type, host strain and selection pressure were analyzed . Using a secretion system based on the controlled expression of the kil gene, the expression of phytase was improved and the enzyme was released into the culture medium at a high level . An effective fermentation strategy based on fed-batch operation was developed. Appl Microbiol Biotechnol, 2002 Sep, 59(6), 679 - 84 Epub 2002 Jul 13. Biotransformation of p-xylene and 2,6-dimethylnaphthalene by xylene monooxygenase cloned from a Sphingomonas isolate; Bramucci M et al.; Sphingomonas strain ASU1 was isolated from an industrial wastewater bioreactor and grew on 2,6-dimethylnaphthalene (2,6-DMN) as the sole carbon/energy source . The genes for a xylene monooxygenase were cloned from strain ASU1 . Expression of the ASU1 xylene monooxygenase was compared to expression of the pWWO xylene monooxygenase in Escherichia coli . Both monooxygenases transformed p-xylene and 2,6-DMN by initially hydroxylating one methyl group . In addition, the ASU1 monooxygenase also hydroxylated the second methyl group on p-xylene and 2,6-DMN whereas the pWWO monooxygenase hydroxylated the second methyl group only on p-xylene . Endogenous E . coli enzymes contributed to further oxidation of the resulting aromatic alcohols to form aromatic carboxylates. Biosci Biotechnol Biochem, 2002 Jul, 66(7), 1473 - 8 Phenotype of hepatocyte spheroids in Arg-GLY-Asp (RGD) containing a thermo-reversible extracellular matrix; Park KH et al.; The spheroid of specific cells is often regarded as the better form in artificial organs and mammalian cell bioreactors for improved cell-specific functions . In this study, freshly harvested primary rat hepatocytes, which had been cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were examined for differentiated morphology and enhanced liver-specific functions as compared to a control set (hepatocytes in single-cell form) . A copolymer of N-isopropylacrylamide (98 mole % in the feed) and acrylic acid (poly(NiPAAm-co-AAc)), and the adhesion molecule, an Arg-Gly-Asp (RGD)-incorporated thermo-reversible matrix, were used to entrap hepatocytes in the form of either spheroids or single cells . In a 28-day culture period, the spheroids in the RGD-incorporated gel maintained higher viability and produced albumin and urea at constant rates, while there was lower cell viability and less albumin secretion by the spheroids in p(NiP