Can J Physiol Pharmacol, 1994 Apr, 72(4), 407 - 14 Digestion and absorption of food: usefulness and limitations of in vitro models; Savoie L; The digestion and absorption of food is a spatiotemporal and dynamic process involving complex enzymatic and transport reactions, and it is illusive to try to reproduce in a single model all these biochemical and physiological events . A more practical and realistic approach is to separately evaluate the specific contributions of oral and gastric digestion, intestinal digestion by pancreatic enzymes, brush-border hydrolysis, and eventually intestinal absorption and enterocyte metabolism . The models proposed must be versatile enough to be able to modify their conditions of operation according to physiological adaptation to food . Enzymatic preparations must be kept close to physiological conditions in regard to their nature and their mode of operation . A digestion cell and a peptidase bioreactor were developed for this purpose . The challenge is to find a way to integrate all these data . This can be partially achieved by selecting techniques that allow the collection and isolation of reaction products from one step for use as substrates for the next event . Various models are presented to illustrate this concept as applied to food protein. Glycoconj J, 1994 Apr, 11(2), 153 - 62 The ganglioside GD1 alpha' IV3Neu5Ac, III6Neu5Ac-GgOse4Cer, is a major disialoganglioside in the highly metastatic murine lymphoreticular tumour cell line MDAY-D2; Muthing J et al.; The aim of the present study was to investigate the ganglioside expression of the highly metastatic murine lymphoreticular tumour cell line MDAY-D2 . Cells were propagated under controlled pH conditions and oxygen supply in bioreactors of 1 and 7.5 l volumes by repeated batch fermentation . Gangliosides were isolated from 2.7 x 10(11) cells, purified by silica gel chromatography and separated into mono- and disialoganglioside fractions by preparative DEAE anion exchange high performance liquid chromatography . Individual gangliosides were obtained by preparative thin layer chromatography . Their structural features were established by immunostaining, fast atom bombardment and gas chromatography mass spectrometry . In addition to gangliosides of the GM1a-pathway (GM2, GM1a and GD1a) and GM1b (IV3Neu5Ac-GgOse4Cer) and GalNAc-GM1b of the Gm1b-pathway, the disialoganglioside GD1 alpha (IV3Neu5Ac, III6Neu5Ac-GgOse4Cer) was found in equal amounts compared to GD1a (IV3Neu5Ac, II3Neu5Ac-GgOse4Cer) . All gangliosides were substituted with C24:0, 24:1 and C16:0 fatty acids, sphingosine and N-acetylneuraminic acid as the sole sialic acid. J Biotechnol, 1994 Mar 31, 33(2), 107 - 22 Unified modeling framework of cell death due to bubbles in agitated and sparged bioreactors; Wang NS et al.; A modeling framework is proposed to assess the detrimental effects of air sparging and other bubble phenomena (vortex entrainment, coalescence, bursting) on freely suspended cells in an aerated, agitated bioreactor . It is assumed that cells may be rendered nonviable by bubble breakup/coalescence within the medium, by bubble formation at the sparger, or by bubble bursting at the free surface . Some plausible mechanisms are argued from the energetic view point . The dominant parameters in each case are the cell-bubble encounter rate and the bubble breakup/bursting rate . These inactivation processes lead to a Michaelis-Menten expression for the specific cell death rate, which is shown to be linearly proportional to the specific bubble interfacial area (total bubble surface area per unit volume of media) . By using published viable cell concentration data for retarded growth of mammalian cells due to sparging, the interfacial area correlation is demonstrated . The method is generalized to aerated bioreactor conditions . The article offers a unique, consistent perspective on how cell death can be viewed. J Biotechnol, 1994 Mar 15, 33(1), 21 - 31 Intracellular calcium response of Sf-9 insect cells exposed to intense fluid forces; Aloi LE et al.; Suspension cell cultures are exposed to periodic high intensity, short duration fluid forces by circulating them through a flow loop containing a capillary, which simulates what a cell experiences in a stirred bioreactor . Sf-9 insect cells exhibit an increase in intracellular calcium concentration, {Ca2+}i when exposed to these cyclic fluid forces . Flow through the capillary spans both the laminar and turbulent regime and the calcium response is not dependent on the transition to turbulence . The calcium response is a nearly linear function of the rate of energy dissipation per mass of fluid in the capillary . The source of the increased calcium ions in the cell cytosol is within the cell itself, indicating that the calcium response is a cellular response to fluid forces and not a matter of increased plasma membrane permeability to Ca2+ ions . Flow cytometry on hydrodynamically stimulated and unstimulated cells reveals that the increase in the intracellular calcium concentration averaged over the cell population is due to an increase in intracellular calcium concentration in only a small sub-population of the entire suspension. Artif Organs, 1994 Mar, 18(3), 226 - 30 Hepatocyte culture between woven capillary networks: a microscopy study; Gerlach J et al.; A multi-compartment capillary membrane culture model with independently perfused three-dimensionally woven capillaries was developed for immobilization of hepatocytes in bioreactors . This enables spatial restructuring of cells and enhanced mass transfer performance with more efficient oxygenation and metabolite exchange . Seeding density defines cell behaviour in this model . With low densities cells attach to the membranes and flatten . Increasing density leads to spontaneous formation of aggregates which are immobilized between the capillaries. Biodegradation, 1994 Mar, 5(1), 1 - 11 Optimization and maintenance of soluble methane monooxygenase activity in Methylosinus trichosporium OB3b; Bowman JP et al.; Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater . A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity in Methylosinus trichosporium OB3b . Methylosinus trichosporium OB3b attained peak sMMO activity (275-300 nmol of naphthol formed h-1 mg of protein-1 at 25 degrees C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium . With the onset of methane limitation however, sMMO activity rapidly declined . It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (mu max 0.08 h-1) and growth yields (0.4-0.5 g cells/g CH4) and near maximal activities of sMMO . In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45-50% of the initial peak level and this was maintained over several weeks . The addition of d-biotin, pyridoxine, and vitamin B12 (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25-75% . The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity. J Chem Technol Biotechnol, 1994 Mar, 59(3), 297 - 302 Residence time distribution in a packed bed bioreactor containing porous glass particles: influence of the presence of immobilized cells; De Backer L et al.; An experimental investigation of the liquid phase residence time distribution (RTD) in a packed bed bioreactor containing porous glass particles is presented . For Re < 1, intraparticle forced convection is negligible and only diffusion, characterized by an effective diffusion coefficient, must be considered to describe the mass transfer process between the extraparticle and the intraparticle fluid phase . For Re > 1, the mass transfer rate becomes dependent on the liquid flow rate, indicating the existence of intraparticle convection . A model including axially dispersed flow for the external fluid phase and an 'apparent' effective diffusivity that combines diffusion and convection, predicts experimental RTD data satisfactorily . Yeast cells immobilized inside the porous glass beads did not affect the mass transfer rate at low biomass loading . At high biomass loading (0.02 g yeast cells g-1 carrier), the mass transfer rate between the extraparticle and intraparticle fluid phase was significantly decreased . Comparison of the RTD data from experiments performed in the presence and absence of cells in the external fluid phase revealed that the mass transfer rate is influenced by the cells immobilized inside the porous particles and not by the cells present in the external fluid phase. Biotechnol Prog, 1994 Mar-Apr, 10(2), 198 - 206 Viable cell recycle with an inclined settler in the perfusion culture of suspended recombinant Chinese hamster ovary cells; Searles JA et al.; The perfusion culture of suspended mammalian cells requires a cell retention device, the best of which will retain all viable cells and reject all nonviable cells and debris . The inclined settler is a passive, simple, inexpensive, and easy-to-maintain device that has been shown in the past to selectively remove single nonviable cells of hybridoma cultures . In this work, we have demonstrated the preferential return of viable recombinant Chinese hamster ovary (CHO) cells through the use of a three-port settler maintained at lower temperatures and vibrated to reduce cell attachment and enhance cell return to the bioreactor . The residence time of CHO cells in the cooled, vibrated settler was determined by flow-cytometric discrimination of tracer recombinant CHO cells . Cells returning to the bioreactor through the underflow had an average residence time of 1.46 h in the settler . During perfusion cultures with cell densities above 10(6) cells/mL, cells seen to be stalled within the settler were easily dislodged by periodic air bubbling using a simple back-flushing procedure in which headspace gas was brought through the settler underflow port . The resuspended cells were returned to the bioreactor within an average of 32 min after bubbling . This study demonstrates that inclined sedimentation technology can be utilized to selectively recycle viable recombinant CHO cells with only a short retention time in an inclined settler. Enzyme Microb Technol, 1994 Mar, 16(3), 253 - 7 Interaction of transport resistances with biochemical reaction in packed-bed solid-state fermentors: effect of temperature gradients; Ghildyal NP et al.; In solid-state fermentation, the interaction of transport phenomena with biochemical reactions has a considerable effect on the productivity of the bioreactor . Previous work on solid-state fermentation in tray fermentors in our laboratory indicated that heat transfer resistance results in steep temperature gradients within the solid substrate bed, which in turn adversely affect the biochemical reaction and enzyme activity . This problem of heat accumulation during the course of fermentation has been alleviated to a considerable extent using a packed-column bioreactor with forced aeration in the present work . Experimental studies were conducted in a packed-column bioreactor utilizing wheat bran as substrate and the fungus Aspergillus niger CFTRI 1105 for the production of the enzyme amyloglucosidase . The enzyme activities were estimated and temperatures were recorded at different bed heights, for different air flow rates during the course of fermentation . The results indicated that the temperature gradients caused by heat transfer resistances were reduced considerably with corresponding increases in enzyme activity. Biotechnology (N Y), 1994 Mar, 12(3), 281 - 4 Acoustic cell filter for high density perfusion culture of hybridoma cells; Trampler F et al.; We have developed a flow-through device which uses high frequency, low energy ultrasonic resonance fields to transiently aggregate hybridoma cells and return them by sedimentation to a perfusion bioreactor . The system retained up to 99 percent of the inflowing viable cells with no measurable effect on viability . Viable cells were selectively retained at up to 3 percent higher efficiency than nonviable cells . A stirred tank bioreactor was operated for 700 hours with acoustic cell recycle . Concentrations greater than 5 x 10(7) cells/ml were attained with a 5-fold increase in antibody concentration and a 70-fold increase in volumetric productivity compared with batch culture. J Biotechnol, 1994 Feb 28, 32(3), 231 - 8 Expression of alpha 1-proteinase inhibitor in Escherichia coli: effects of single amino acid substitutions in the active site loop on aggregate formation; Schulze AJ et al.; Overproduction of eukaryotic proteins in microorganisms often leads to the formation of insoluble protein aggregates which accumulate as intracellular inclusion bodies . alpha 1-Proteinase inhibitor (alpha 1-PI) when produced as a cytoplasmic protein in Escherichia coli (E . coli) forms inclusion bodies containing the majority of the inhibitor in an inactive form . Several variants of alpha 1-PI with single amino acid substitutions within their active site loop (amino acids 345-358) were produced in a bioreactor showing that substitution of Met351 with Glu resulted in significantly reduced aggregate formation compared to the other variants and to wild-type protein . In addition, this variant proved to be fully functional as a proteinase inhibitor . Based on these findings and on results of previous structural studies a mechanism for aggregate formation during expression of alpha 1-PI is suggested. J Biotechnol, 1994 Feb 14, 32(2), 127 - 38 Optical triple sensor for measuring pH, oxygen and carbon dioxide; Weigl BH et al.; A triple sensor unit consisting of opto-chemical sensors for measurement of pH, oxygen and carbon dioxide in bioreactors is presented . The pH and the CO2 sensor are based on the color change of a pH-sensitive dye immobilized on a polymeric support . The resulting changes in absorption are monitored through optical fibers . The oxygen sensor is based on the quenching of the fluorescence of a metal-organic dye . All three sensors are fully LED compatible . The sensitive membranes consist of plastic films and can be stored and replaced conveniently . The sensors are sterilizable with hydrogen peroxide and ethanol . In addition, the pH sensor is steam sterilizable . Accuracy, resolution and reproducibility fulfill the requirements for use in biotechnological applications . Calibration procedures for each sensor are presented . The working principle and the performance of all three sensors are described, with particular emphasis given to their application in bioreactors. Appl Biochem Biotechnol, 1994 Feb, 44(2), 151 - 60 Immobilized carboxypeptidase N . A potent bioreactor and specific adsorbent for peptides; Wang W et al.; Carboxypeptidase N-Sepharose was prepared by covalent immobilization of purified human plasma carboxypeptidase N . More than 98% of the carboxypeptidase N was immobilized; 42% of the applied activity can be detected on the support . The column has excellent capabilities to quantitatively remove carboxy-terminal basic amino acids from peptides, as is demonstrated using the synthetic peptide substrate hippuryl-L-arginine and the nonapeptide bradykinin, and remains stable for several months . In contrast with apocarboxypeptidase B-Sepharose, apocarboxypeptidase N-Sepharose poorly binds its substrates. Enzyme Microb Technol, 1994 Feb, 16(2), 151 - 8 A new enzyme immobilization procedure using copper alginate gel: application to a fungal phenol oxidase; Palmieri G et al.; A new procedure was developed for enzyme immobilization by entrapment in copper alginate gel . The mechanical properties of the copper alginate gel were characterized and compared with those of the most widely used calcium alginate . The system was applied to the immobilization of a fungal phenol oxidase . Optimal conditions for enzyme immobilization were set up: the system immobilized 85% of the enzyme, and the remaining 15% was recovered in the aqueous immobilization medium . The stability and activity of the immobilized enzyme were studied . After immobilization, the enzyme was active in a wider pH range, the temperature of its optimal activity was shifted to lower values, and the possibility of storage at 4 degrees C was greatly improved . The immobilized enzyme generally increased the rate of oxidation of various substrates . The results indicate a potential use of this system for the construction of bioreactors to be used in the detoxification of polluted waste waters. J Immunol Methods, 1994 Jan 3, 167(1-2), 109 - 19 Enhanced IgG production in eRDF media with and without serum . A comparative study; Chua F et al.; The performance of three basal media RPMI, DMEM/F12 (DF) and eRDF (enhanced RDF, RPMI:DMEM:F12 in 2:1:1) were evaluated in cultures with and without serum with respect to cell proliferation, metabolism and monoclonal antibody (Mab) productivity . Based on the ease of adaptation, growth rate, maximum cell density and Mab production, the media were ranked as follows: eRDF > DF > RPMI . This was true for serum-free (SF) and serum supplemented (SS) media in static and shaker cultures . Growth performances in static and shaker cultures were consistently 20-50% lower in all three SF media compared to the corresponding SS conditions . Antibody titres in DF/SF and RPMI/SF cultures, irrespective of the culture condition, were generally similar or slightly lower than their SS counterparts . However, eRDF/SF medium yielded a much higher Mab titre (193 mg l-1) compared to eRDF/SS medium (145 mg l-1) . This was also six times higher than the lowest titre of 30 mg l-1 in RPMI/SF medium . Hybridomas in eRDF/SF were further adapted to media without bovine serum albumin (eRDF/SF-BSA) . Maximum cell densities in these cultures improved with scale up, from 1.1 x 10(6) ml-1 in static, to 1.9 x 10(6) ml-1 in shaker flasks, to 2.5 x 10(6) ml-1 in bioreactors . However, Ig levels remained between 100-130 mg l-1 which were much lower than in eRDF/SF medium . Thus BSA appears to be necessary for Ig production . The manufacturing cost (excluding purification) of Ig using eRDF was calculated to be between 17-50% of the price of the other two media and therefore this is regarded as the best medium for Ig production. In Vitro Cell Dev Biol Anim, 1994 Jan, 30A(1), 23 - 9 Primary cultures of rat hepatocytes in hollow fiber chambers; Jauregui HO et al.; Hepatocyte culture may represent an alternative to the use of animals to study drug detoxification by the liver . An ideal in vitro system should closely mimic the in vivo environment by providing continuous media perfusion and oxygenation, and should facilitate sampling of cells and culture media . To meet these criteria, a hollow fiber bioreactor seeded with isolated rat hepatocytes was developed and tested by measuring the formation of three products of the oxidative metabolism of diazepam and the glucuronidation of phenolsulfonphthalein (PSP) . To compare the performance of conventional monolayer culture to that of the bioreactor system, diazepam metabolism was studied for 45 days in both systems . The oxygen dependency of diazepam metabolism was evaluated by perfusing the bioreactor in an oxygen-rich atmosphere (30%) . Total diazepam metabolism was twofold higher in the O2-rich perfused hollow fiber cultures than in the cultures perfused under normal conditions, reflecting an increase in temazepam and oxazepam production . Diazepam detoxification activity was significantly enhanced by oxygen (P < or = 0.001) over the life of the perfused cultures . PSP metabolism was similar in all three culture systems . By Day 10, diazepam metabolism in the oxygenated bioreactor system was 44% of the in vivo activity of rat hepatocytes . This activity dropped to 30% by Day 25 of culture . These results justify the use of perfused culture systems for in vitro detoxification studies as an alternative to animal use and emphasize the capacity of a culture device perfused under O2-enriched conditions to maintain long-term P450 activity of rat hepatocytes. Hum Gene Ther, 1994 Jan, 5(1), 19 - 28 Improved methods of retroviral vector transduction and production for gene therapy; Kotani H et al.; To facilitate clinical applications of retroviral-mediated human gene transfer, retroviral vectors must be of high titer and free of detectable replication-competent retroviruses . The purpose of this study was to optimize methods of retroviral vector production and transduction . Studies were conducted using 22 retroviral vector producer cell lines . Inactivation of retroviral vectors was greater at 37 degrees C than at 32 degrees C . A 5- to 15-fold increase of vectors was produced at 32 degrees C compared to 37 degrees C; the vector increase at 34 degrees C was intermediate . For example, PA317/G1Na.40 grew to a titer of 1.8 x 10(7) cfu/ml at 32 degrees C, compared to 5.0 x 10(5) cfu/ml at 37 degrees C . The production of retroviral vectors was scalable achieving similar results in flasks, roller bottles, or a CellCube Bioreactor . Retroviral vectors were concentrated 15-24 times with vector recovery ranging from 91 to 96% in a Pellicon tangential flow filtration system . Retroviral supernatants were successfully lyophilized . The combination of glucose or sorbitol with gelatin resulted in recovery rates of 64-83% . In studies on transduction by retroviral vectors, centrifugation of vector supernatants onto target cells significantly increased transduction efficiency as measured by vector titration for G418 resistance, fluorescence-activated cell sorting (FACS), and polymerase chain reaction (PCR) analyses . The combination of the above methods has significantly increased the growth and transduction by this vector system. Res Microbiol, 1994 Jan, 145(1), 49 - 52 Disposal of slop oil and sludges by biodegradation; Jack TR et al.; These pilot tests indicate that immobilized microbial populations can degrade a wide range of crude oils absorbed into a properly prepared peat matrix with surprising speed and flexibility . Depending on the hydrocarbon composition, ultimate disposal by landfarming or landfilling of the residues is possible . In the former case, performance of the landfarm will be enhanced and environmental concerns related to its operation reduced . In terms of ease of operation, capital and operating costs and reduced environmental concerns, the novel bioreactor presented here meets the requirements and cost constraints associated with on-site slop oil and sludge disposal for the sorts of volumes normally encountered . A patent has been applied for (Francis and Jack, 1991) . The technological issues in this development process largely arose from requirements and constraints associated with the target application . Scientific issues surrounding the enhanced biodegradation, seen especially for absorbed heavy oil, remain unresolved . These may or may not be picked up in further development and optimization as need and cost allow. Crit Rev Biotechnol, 1994, 14(2), 75 - 107 Immobilization of cells for application in the food industry; Groboillot A et al.; Immobilization of cells offers advantages to the food process industries, including enhanced fermentation productivity and cell stability and reduced downstream processing costs due to facilitated cell recovery and recycle . This article summarizes the varied immobilization methodologies, including adsorption, entrapment, covalent binding, and microencapsulation . Examples of interest to the food industry are provided, together with a review of the physiological effects of immobilization . Topics in process engineering include immobilized cell bioreactor configurations and the scale-up potential of the various immobilization techniques. Zentralbl Chir, 1994, 119(5), 334 - 40 {Cell culture model for hepatocyte culture in bioreactors for metabolic utilization in hybrid liver support system}; Gerlach J et al.; Hybrid systems with hepatocyte cultures in bioreactors are in development for therapeutical liver assistance . Here, a culture model with a special bioreactor construction was developed: Capillary membrane systems create a three dimensional artificial framework for hepatocyte adhesion, aggregation and reorganization of tissue structures . Many of small parallel capillary membrane units perfuse a few hepatocytes . Different capillary materials enable different functions . Cell perfusion between independent plasma inflow and outflow capillaries, independent oxygen supply and carbon dioxide removal as well as a co- culture with sinusoidal endothelial cells are possible . An in vitro study with bioreactors (n = 9), containing 2.5 x 10(9) pig hepatocytes was performed, measuring external metabolism of the cells: cytochrome P450-activity (midazolam metabolism, lidocaine/MEGX-test), synthesis (albumin), liver function test (galactose elimination) and cell alteration (LDH, GOT, GLDH, GPT, gamma GT) were investigated . The results demonstrate that external function of primary hepatocytes can be maintained over a period of at least three weeks. Biochem Mol Biol Int, 1994 Jan, 32(1), 87 - 94 Interaction of cationic phospholipid vesicles with carbonic anhydrase; Annesini MC et al.; The possibility of entrapping the enzyme carbonic anhydrase into liposomes, in order to obtain small, membrane-confined bioreactors for biotechnological or biomedical applications, was studied . Neutral liposomes (dipalmitoylphosphatidylcholine/cholesterol) or cationic liposomes (dipalmitoylphosphatidylcholine/cholesterol/stearylamine) with different dipalmitoylphosphatidylcholine/stearylamine ratios have been used to trap carbonic anhydrase . Kinetic experiments showed that carbonic anhydrase was being trapped into cationic liposomes, but not into neutral ones . A significant amount of carbonic anhydrase was sitting onto the external surface of liposomes when the ratio dipalmitoylphosphatidylcholine/cholesterol/stearylamine was 6:3:1, but not when it was 5:3:2 . Morphological analysis by electron microscopy showed that the presence of carbonic anhydrase induced a significant swelling in the 6:3:1 cationic vesicles, related to the activity of the enzyme. Bioelectromagnetics, 1994, 15(4), 303 - 13 Effect of microwave radiation on the permeability of carbonic anhydrase loaded unilamellar liposomes; Orlando AR et al.; The influence of 2.45 GHz microwave exposure (6 mW/g) on the diffusion processes in enzyme-loaded unilamellar liposomes as bioreactors was studied . The enzyme carbonic anhydrase (CA) was entrapped into cationic unilamellar vesicles . Previous kinetic experiments showed a very low self-diffusion rate of the substrate p-nitrophenyl acetate (PNPA) across intact liposome bilayer . A twofold increase in the diffusion rate of PNPA through the lipid bilayer was observed after 120 min of microwave radiation compared to temperature control samples . The microwave effect was time dependent . The enzyme activity, as a function of increased diffusion of PNPA, rises over 120 min from 22.3% to 80% . The increase in stearylamine concentration reduces the enzyme activity from 80% to 65% at 120 min . No enzyme leakage was observed. Physiol Res, 1994, 43(2), 137 - 9 Adrenergic lipolysis in immobilized and perfused adipocytes; Lincova D et al.; The method of cellular immobilization and perfusion was applied to adipocytes . The lipolytic effect of isoprenaline, whose action is produced as a result of receptor-drug interaction, was followed . An agarose solution kept at at 37 degrees C was mixed 1:1 with the cell suspension . Thereafter, adipocytes were immobilized in the agarose threads . The lipolytic effect of 0.1 ml of isoprenaline (1 x 10(-4) mol/l), that was rapidly introduced to the cell perfusion inlet in a non-recirculating system, was monitored by assessing glycerol production . The immobilized and perfused adipocytes exhibited significant lipolytic activity . After reaching the maximum effect, 0.1 ml of propranolol (1 x 10(-3) mol/l) that was applied to the bioreactor inlet, abolished the isoprenaline effect . The present data demonstrate the potential applicability of immobilized perfused adipocytes for various kinds of studies. Physiol Res, 1994, 43(2), 131 - 5 A preliminary evaluation of drug biotransformation in hepatocytes of genetically defined rat strains; Hynie S et al.; This study was directed to use the genetically developed isoprenaline-sensitive (S), isoprenaline-resistant (R) and spontaneous hypertensive rats (SHR) as standard diseased animal models for in vitro liver function evaluation of drug biotransformation . Hepatic hexobarbital hydroxylase and glutathione transferase (GST) were evaluated by using hexobarbital and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates, at concentrations of 0.21 mmol/l and 1 mmol/l, respectively . The assay was conducted by using isolated hepatocytes in suspension and hepatocytes in a bioreactor configuration . The data demonstrate that there are certain cellular pharmacokinetic differences in hexobarbital hydroxylase and GST activities in hepatocytes obtained from Wistar, SHR, R and S strains which can be better demonstrated, when using the model of perfused and immobilized hepatocytes. Physiol Res, 1994, 43(2), 127 - 30 Application of the hepatocytes bioreactor to xenobiotic biotransformation; Kamenikova L et al.; This study deals with the application of the previously developed immobilized and perfused isolated hepatocytes as a cellular system for the study of representative phase I and phase II of biotransformation reactions . To illustrate phase I reactions, aminopyrine (0.17-4.25 mmol/l) and hexobarbital (0.2 mmol/l) were selected . For phase II reactions, glutathione transferase activity was evaluated by using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate (0.125-2.0 mmol/l) . Formaldehyde, that was formed from aminopyrine, increased steadily in the perfusion medium with time . The perfused hepatocytes eliminated hexobarbital at a much higher rate than the hepatocytes in suspension . At several time points the amount of CDNB-glutathione conjugate formed per one million hepatocytes in the bioreactor was almost twice the amount formed by the hepatocytes in suspension . The present data illustrate the successful application of the hepatocyte bioreactor in phase I and phase II of xenobiotic metabolism and indicate that the cells were metabolically more active than the cells in suspension. Physiol Res, 1994, 43(2), 121 - 5 Preparation of functionally active immobilized and perfused mammalian cells: an example of the hepatocyte bioreactor; Farghali H et al.; In the present study, a method has been employed for hepatocyte immobilization in agarose threads which allows for cell perfusion . The rat hepatocytes are isolated from the liver . A 1.8% low-gelling agarose solution is prepared in warm Krebs-Henseleit solution . The agarose solution is mixed 1:1 with the hepatocytes and the cells are immobilized in agarose threads by extruding the agarose-cell mixture through cooled Chemfluor teflon (TFE) tubing . Light and electron microscopy studies indicated the integrity of the hepatocytes in the gel matrix . This system allows for liver cell perfusion and viability studies to be carried out non-invasively on the cells and provides data that are comparable to those obtained with a perfused isolated liver . Immobilized hepatocytes are an in vitro system worthy of further evaluation which may be useful in the studies of liver cell metabolism and the response of the liver to foreign chemicals. Physiol Res, 1994, 43(2), 117 - 20 The concept of application of immobilized and perfused mammalian cells (a bioreactor model) in biomedical research; Farghali H et al.; An overview of the concept of cellular immobilization and perfusion as a small laboratory bioreactor model is presented . The cellular systems currently used may be described as static . This is due to conditions of hypoxia and waste product build-up that affect cell physiology . Cellular immobilization and perfusion is, therefore, expected to maintain the cells for very long periods of time under approximately physiological conditions . A number of applications of immobilized perfused hepatocytes and other cellular systems such as adipocytes and Sertoli cells are described in addition to various other cell lines . Moreover, it is suggested that the bioreactor may have potential use as a bioartificial organ. Biomed Pharmacother, 1994, 48(7), 282 - 6 Endosome-lysosomes and neurodegeneration; Mayer RJ et al.; A number of the major human and animal neurodegenerative diseases, such as Alzheimer's disease and sheep scrapie, are characterised by deposits of amyloid, arising through incomplete breakdown of membrane proteins . Although our knowledge concerning these diseases is increasing, they remain largely untreatable . Recently, attention has focussed on the mechanisms of production of different types of amyloid and the likely involvement within cells of acid compartments called endosome-lysosomes . These organelles may be 'bioreactor' sites for the unfolding and partial degradation of membrane proteins to generate the amyloid materials . These subsequently become expelled from the cell, or are released from dead cells, and accumulate as pathological entities . Common features of the disease processes give new direction to therapeutic intervention. J Hematother, 1994 Fall, 3(3), 213 - 8 Future paradigm for autologous bone marrow transplantation: tumor purging and ex vivo production of normal stem and progenitor cells; Rummel SA et al.; A major concern in autologous bone marrow transplantation (ABMT) is the possible contamination of the graft with tumor cells . Transplantation of malignant cells, along with normal hematopoietic stem and progenitor cells, may contribute to relapse of disease . Therefore, a growing strategy is to subject autologous marrow to some type of purging procedure to eliminate tumor cells selectively . Transplantation of purged marrow, however, often results in a delayed engraftment associated with (specific or nonspecific) loss of normal stem and progenitor cells during manipulations related to the purging process . A new and burgeoning field in the area of clinical bone marrow transplantation is the ex vivo production of stem and progenitor cells . Several advantages accrue to this strategy . First, this technology makes it possible to expand the stem and progenitor cell population of a small volume of bone marrow or mobilized peripheral blood (MPB), thus lessening the initial tumor burden to be purged . Secondly, ex vivo marrow or MPB expansion may overcome the significant problem of delayed engraftment by rebuilding the numbers of normal stem and progenitor cells necessary for both early and durable engraftment . To accomplish these and other objectives, an automated and closed, clinical-scale bioreactor system, based on continuous perfusion technology, is being developed and will soon enter clinical trials. Cancer Biother, 1994 Fall, 9(3), 211 - 24 Growth of tumor derived activated T-cells for the treatment of cancer; Lewko WM et al.; This report describes the production of Tumor Derived Activated Cell cultures (TDAC, also called tumor infiltrating lymphocytes) from patient tumor biopsies and our preliminary experience growing these cells to therapeutic levels using artificial capillary bioreactor cultures . TDAC were successfully grown in medium containing Interleukin 2 from 80% of the 113 tumor biopsies tested . There was no significant difference in success (growth to 1 x 10(9) cells) comparing primary and metastatic tumors . Many of the tumors were shipped to the laboratory from distant sites . Success rate did decrease with the length of time for tumor transport . Interleukin 4 was beneficial in the development of 1 of 4 TDAC cultures which did not grow with IL-2 only . Seventy-seven bioreactor cultures were initiated for 31 patients . On the average, 1.9 x 10(9) TDAC were inoculated per bioreactor; 3.3 x 10(10) were harvested in 22 days . Twelve liters of medium were required per 1 x 10(10) TDAC produced . TDAC cultures contained T cells with variable ratios of CD4 to CD8 cells . Secreted granulocyte monocyte colony stimulating factor, interferon gamma and tumor necrosis factor were measured in the bioreactor cartridge conditioned medium . Twenty three patients were evaluated . Partial responses were observed in 4 patients including a dramatic remission of scalp nodules in a patient with renal cancer . Results showed that therapeutic amounts of TDAC cells may be produced in a reasonable and cost effective manner using artificial capillary bioreactor cultures. Cytotechnology, 1994, 15(1-3), 351 - 63 Towards the development of a bioartificial pancreas: immunoisolation and NMR monitoring of mouse insulinomas; Sambanis A et al.; A promising method for diabetes treatment is the implantation of immunoisolated cells secreting insulin in response to glucose . Cell availability limits the application of this approach at a medically-relevant scale . We explore the use of transformed cells that can be grown to large homogeneous populations in developing artificial pancreatic tissues . We also investigate the use of NMR in evaluating, non-invasively, cellular bioenergetics in the tissue environment . The system employed in this study consisted of mouse insulinoma beta TC3 cells entrapped in calcium alginate/poly-L-lysine (PPL)/alginate beads . The PPL layer imposed a molecular weight cutoff of approximately 60 kDa, allowing nutrients and insulin to diffuse through but excluding high molecular weight antibodies and cytotoxic cells of the host . We fabricated a radiofrequency coil that can be double-tuned to 1H and 31P, and an NMR-compatible perfusion bioreactor and support circuit that can maintain cells viable during prolonged studies . The bioreactor operated differentially, was macroscopically homogeneous and allowed the acquisitions of 1H images and 31P NMR spectra in reasonable time intervals . Results indicated that entrapment had little effect on cell viability; that insulin secretion from beads was responsive to glucose; and that the bioenergetics of perfused, entrapped cells were not grossly different from those of cells never subjected to the immobilization procedure . These findings offer promise for developing an artificial pancreatic tissue for diabetes treatment based on continuous cell lines. Cytotechnology, 1994, 15(1-3), 321 - 8 Quantitative investigations of cell-bubble interactions using a foam fractionation technique; Tan WS et al.; Previous work by the authors and others has shown that suspended animal cell damage in bioreactors is caused by cell-bubble interactions, regardless whether the bubbles are from bubble entrainment or direct gas sparging . As approach to measure the adsorptivity of animal cells to bubbles, a modified batch foam fractionation technique has been developed in this work and proven to be applicable . By using this technique, the number of cells absorbed per unit bubble surface area and the adsorption coefficients have been measured to quantify hybridoma cell-bubble interactions, and the preventive effects of serum and Pluronic F68 on these interactions . It was demonstrated quantitatively that the hybridoma cells adhere to bubbles spontaneously and significant numbers exist in the foam, and that both the serum and Pluronic F68 provide strong prevention to these cell-bubble interactions . The results obtained provide criteria for bioreactor operation and medium formulation to prevent cell-bubble interactions and cell damage in the culture processes. Cytotechnology, 1994, 15(1-3), 311 - 20 Cells and bubbles in sparged bioreactors; Chalmers JJ; Ever since animal cells have been grown in-vitro, various techniques have been used to supply the cells with oxygen . The most simple and commonly used 'large-scale' technique to provide oxygen is through the introduction of gas bubbles . However, almost since the beginning of in-vitro cell culture, empirical observations have indicated that bubbles can be detrimental to the cells . This review will discuss the background of the problem, review the relevant research on the topic, attempt to provide a coherent summary of what we know from all of this research, and finally outline what still needs to be investigated . Specific topics to be covered include: experimental correlations of cell damage with bubbles, cell attachment to bubbles, the hydrodynamics of bubble rupture, bioreactor studies, visualization studies, and computer simulations and qualification of cell death as a result of bubble rupture. Cytotechnology, 1994, 15(1-3), 301 - 9 Design of a bubble-swarm bioreactor for animal cell culture; Gudermann F et al.; A stationary bubble-swarm has been used to aerate a mammalian cell culture bioreactor with an extremely low gas flow rate . Prolonging the residence time of the gas bubbles within the medium improved the efficiency of the gas transfer into the liquid phase and suppressed foam formation . An appropriate field of speed gradients prevented the bubbles from rising to the surface . This aeration method achieves an almost 90% transfer of oxygen supplied by the bubbles . Consequently, it is able to supply cells with oxygen even at high cell densities, while sparging with a gas flow of only 0.22 x 10(-3) -1.45 x 10(-3) vvm (30-200 ml/h) . The reactor design, the oxygen transfer rates and the high efficiency of the system are presented . Two repeated batch cultures of a rat-mouse hybridoma cell line are compared with a surface-aerated spinner culture . The used cell culture medium was serum-free, either with or without BSA and did not contain surfactants or other cell protecting agents . One batch is discussed in detail for oxygen supply, amino acid consumption and specific antibody production. Cytotechnology, 1994, 15(1-3), 3 - 9 Cultural and physiological factors affecting expression of recombinant proteins; Griffiths JB et al.; The variability in expression of recombinant proteins has been analyzed with regard to (a) comparison of clones from the same transfection experiment; (b) comparison of the same genetic construct in different cell lines; (c) the effect of the culture system used (free suspension, aggregate suspension, and microcarrier); and (d) physiochemical parameters in long-term (100d) culture in a macroporous fixed bed bioreactor (FBR) . Differences in product expression between clones were accompanied by differences in growth rates, metabolic kinetics, and ability to grow in suspension as opposed to attached culture . The single most important factor affecting product expression when comparing constructs (for SEAP and IgG), cell lines (BHK 21 and myeloma), and culture systems was whether cells were grown in an attached or suspension mode . Thus key factors could be related to cell morphology (suspension versus monolayer), the presence of microenvironments and physiological stress to control growth rate . The relationship of key process parameters to volumetric and specific rAb productivity of the FBR was investigated in a partial factorial experiment with a rBHK cell line . The highest productivity levels are associated with a combination of the highest values tested for re-cycle (195 ml min-1) and dilution rates (1 d-1) and glutamine concentration (2.5 mmol 1-1), plus the lowest values for bead size (2 mm) and inoculum density (10(7) m1-1) . Together with data from fluidised bed cultures, these results suggest that higher productivity is not primarily the result of greater cell numbers within the system but more the physicochemical definition of the system. Cytotechnology, 1994, 15(1-3), 271 - 9 A direct computer control concept for mammalian cell fermentation processes; Buntemeyer H et al.; In the last 10 years, new assignments and the special demands of mammalian cells to the culture conditions caused the development of complex small scale fermentation setups . The use of continuous fermentation and cell retention devices requires appropriate process control systems . An arrangement for control and data-acquisition of complex laboratory-scale bioreactors is presented . The fundamental idea was the usage of a standard personal computer, which is connected to pumps, valves and sensors via ADA-transformation . The possibility of free programming allowed the development of user-oriented software, especially designed for the far-reaching requirements of a university laboratory in the field of animal cell culture . Control of aeration, pumps, data-acquisition and data-storage are combined within one program, which allows the automation of standard operations like measurement of kLa- or OTR-values . Pump control algorithms for all common fermentation strategies (batch, fed batch, chemostat, perfusion) are included and can be selected any time during cultivation . Oxygen partial pressure and pH are controlled via direct digital control (ddc), providing simple adaption of control parameters and set points to current fermentation conditions. Cytotechnology, 1994, 15(1-3), 253 - 8 Vortex flow filtration of mammalian and insect cells; Hawrylik SJ et al.; The use of vortex flow filtration for harvesting cells or conditioned medium from large scale bioreactors has proven to be an efficient, low shear method of cell concentration and conditioned medium clarification . Several 8-10 L batches of the human histiocytic lymphoma U-937 cell line (ATCC CRL 1593) were concentrated to less than 1 L by vortex flow filtration through a 3.0 microns membrane . An aggressive filtration regimen caused a 17% loss of cell viability and a 32% loss of IL-4 receptor binding capacity when compared to a batch centrifuged control . A reduction of the rotor speed from 1500 to 500 RPM and reduction of system back pressure from 10 to 0 PSIG resulted in cell viability and IL-4 binding capacity comparable to the control . Several 10 L batches of baculovirus infected Sf-9 cells were also concentrated to less than 1 L by vortex flow filtration through a 3.0 microns membrane . SDS-PAGE analysis of filtrate samples showed that aggressive filtration caused cell damage which led to contamination of the process stream by cellular lysate . When rotor speed was reduced to 500 RPM and system back pressure was reduced to 0 PSIG, the amount of contaminating lysate proteins in filtrate samples was comparable to a batch centrifuged control. Cytotechnology, 1994, 15(1-3), 177 - 86 Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells; Hensler WT et al.; The baculovirus infection process of Spodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinant Autographa californica (AcNPV) virus expressing beta-galactosidase enzyme as a model system . A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection . The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident . Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection . The optimal harvest time varied, depending on which technique--SDS-PAGE, chromogenic (ONPG) or fluorometric (C12FDG)--was used to monitor beta-galactosidase production . Specific beta-galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units beta-galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities. Cytotechnology, 1994, 15(1-3), 17 - 29 Applications of improved stoichiometric model in medium design and fed-batch cultivation of animal cells in bioreactor; Xie L et al.; In our previous work (Xie and Wang, 1994a), a simplified stoichiometric model on energy metabolism for animal cell cultivation was developed . Fed-batch experiments were performed in T-flasks using this model in supplemental medium design (Xie and Wang, 1994b) . In this work, the major pathways of glucose and glutamine metabolism were incorporated into the stoichiometric model . Fed-batch culture was conducted in a 2-liter bioreactor with appropriate process control strategies . Nutrient concentrations, especially glucose and glutamine, were maintained at constant but low levels through the automated feeding of a supplemental medium formulated using the improved stoichiometric model . The formation of toxic byproducts, such as ammonia and lactate (Hassell et al., 1991), was greatly reduced . The specific lactate production rate was decreased by 62-fold compared with batch culture in bioreactor and by 8-fold compared to fed-batch culture in T-flask using the previous stoichiometric model . Ammonia formation was also decreased compared with both the batch and fed-batch cultures . Most importantly, the monoclonal antibody concentration reached 900 mg l-1, an increase of 17- and 1.6-fold compared with the batch and fed-batch cultures respectively. Cytotechnology, 1994, 15(1-3), 157 - 67 Relationship between oxygen uptake rate and time of infection of Sf9 insect cells infected with a recombinant baculovirus; Wong TK et al.; Oxygen uptake rates (OUR) of Sf9 insect cells propagated in a serum-free medium (SF900II, Gibco) and of cells infected with a recombinant AcNPV were investigated before and after infection in a laboratory-scale bioreactor . The volumetric OURs of uninfected and exponentially growing cells were found to be proportional to the cell density . For infected cultures, the specific OUR of cells increased immediately after addition of virus and a maximum of 1.3 times the value of uninfected cells was noted for all the cultures between 8 to 30 hours post infection, which coincides with the period at which most viral replication and the majority of DNA synthesis takes place . It was observed that the rate of rise in the specific OUR decreased as the cell density at the time of infection increased, which meant that the later the infection, the later the maximum sOUR was observed . We therefore suggest that OUR measurement can be used to reflect the efficiency of a batch infection . Carbohydrate and amino acid consumption rates from an infected run were analysed in an effort to identify substrate(s) that may be used at increased rates to fuel the rise in oxygen demand observed early in the infection cycle . No observable rise in the consumption rates of glucose or glutamine, which are the major energy sources for animal cells, were seen after infection but an increase in the consumption rates of some amino acids suggests that infected Sf9 cells may utilise amino acids at an enhanced rate for energy post infection. Cytotechnology, 1994, 15(1-3), 145 - 55 Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells; Garnier A et al.; Human 293S cells, a cell line adapted to suspension culture, were grown to 5 x 10(6) cells/mL in batch with calcium-free DMEM . These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content . Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5 x 10(5) cells/mL . In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C, how product yield can be maintained at high cell densities of 2 x 10(6) cells/mL by a medium replacement strategy . This allows the production of as much as 90 mg/L of active recombinant protein per culture volume . Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM . Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system . The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses. Cytotechnology, 1994, 15(1-3), 117 - 28 Induction of apoptosis in oxygen-deprived cultures of hybridoma cells; Mercille S et al.; It is now well documented that apoptosis represents the prevalent mode of cell death in hybridoma cultures . Apoptotic or programmed cell death occurs spontaneously in late exponential phase of batch cultures . Until lately, no specific triggering factors had been identified . Recently, we observed that glutamine, cystine or glucose deprivation induced apoptosis in both hybridoma and myeloma cell lines whereas accumulation of toxic metabolites induced necrotic cell death in these cells . Other triggering factors such as oxygen deprivation might also be responsible for induction of apoptosis . In the present study, induction of cell death by exposure to anoxia was examined in batch culture of the SP2/0-derived hybridoma D5 clone . The mode of cell death was studied by morphological examination of acridine orange-ethidium bromide stained cells in a 1.5 L bioreactor culture grown under anoxic conditions for 75 hours . Under such conditions, viable cell density levelled off rapidly and remained constant for 25 hours . After 45 hours of anoxia, cell viability had decreased to 30% and the dead cell population was found to be 90% apoptotic . In terms of cellular metabolism, anoxia resulted in an increase in the utilization rates of glucose and arginine, and in a decrease in the utilization rate of glutamine . The lactate production rate and the yield of lactate on glucose increased significantly while the MAb production rate decreased . These results demonstrate that glycolysis becomes the main source of energy under anoxic conditions . Cells incubated for 10 hours or less under anoxic conditions were able to recuperate almost immediately and displayed normal growth rates when reincubated in oxic conditions whereas cells incubated for 22 hours or more displayed reduced growth rates . Nonetheless, even after 22 h or 29 h of anoxia, cells reincubated in oxic conditions showed no further progression into apoptosis . Therefore, upon removal of the triggering signal, induction of apoptosis ceased. Cytotechnology, 1994, 15(1-3), 111 - 6 Low temperature cultivation--a step towards process optimisation; Weidemann R et al.; Adherent recombinant BHK cells were cultivated at temperatures between 30 and 37 degrees C . Batch and repeated-batch-cultivations in a 2-litre bioreactor showed a significant influence on metabolism and cell growth . The low-temperature-cultivations showed a lower growth rate and a lower glucose consumption rate and, therefore, less lactate production . On the other hand, the maximum cell density and productivity seemed not to be affected by the temperature reduction. Cytotechnology, 1994, 16(1), 51 - 8 Hybridoma cells in a protein-free medium within a composite gel perfusion bioreactor; Shen BQ et al.; A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels . The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper . During the continuous operation of this system, the total cell density reached 3.9 x 10(7) cells per ml of beads (viability 79.6%) . The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 micrograms per 10(6) viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium . Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed . Leaching of materials from the beads was evident and the major fraction of released materials was alginate. Cytotechnology, 1994, 14(3), 219 - 32 Methods and strategies available for the process control and optimization of monoclonal antibody production; Fu P et al.; The objective of this paper is to explore the range of methods and strategies available for the process control and optimization of monoclonal antibody production by hybridoma cell culture . Emphasis will be placed on the choice of the level of complexity incorporated into the process control and optimisation procedure . It will be shown that the behaviour of hybridomas in culture is influenced by sophisticated cellular metabolic activities and various interactive environmental factors and that the understanding and modelling of the way hybridomas grow in the bioreactor should enable optimisation of bioreactor operating conditions to achieve maximum monoclonal antibody formation . However, due to the lack of on-line instrumentation of important biological variables and the incomplete knowledge of hybridoma cultivation process, there exist many limitations and challenges to the advent of applications of process control and optimisation in this field . To solve the problem, introduction of industrially practical biological measurements and development of new control concepts are inevitable . At the end of this paper, we shall discuss possible schemes for the control of the physiological state of cells in order that balanced cell growth and maximum monoclonal antibody synthesis may be achieved. Cytotechnology, 1994, 14(3), 183 - 90 High density culture of mammalian cells with dynamic perfusion based on on-line oxygen uptake rate measurements; Kyung YS et al.; In a continuous culture with cell retention the perfusion rate must be adjusted dynamically to meet the cellular demand . An automated mechanism of adjusting the perfusion rate based on real-time measurement of the metabolic load of the bioreactor is important in achieving a high cell concentration and maintaining high viability . We employed oxygen uptake rate (OUR) measurement as an on-line metabolic indicator of the physiological state of the cells in the bioreactor and adjusted the perfusion rate accordingly . Using an internal hollow fiber microfiltration system for total cell retention, a cell concentration of almost 10(8) cells/mL was achieved . Although some aggregates were formed during the cultivation, the viability remained high as examined with confocal microscopy after fluorescent vital staining . The results demonstrate that on-line OUR measurement facilitates automated dynamic perfusion and allows a high cell concentration to be achieved. Cytotechnology, 1994, 14(3), 167 - 75 Use of on-line gas analysis to monitor recombinant mammalian cell cultures; Lovrecz G et al.; The development of a system capable of accurately measuring the oxygen uptake and carbon dioxide production rates during mammalian cell cultures is described . A detailed study of the specifications of the various components used in the system for the measurement of gas flow rates and composition, coupled with the validation of the system independent of the bioreactor was carried out . The aim of this study was to identify and eliminate where possible the errors controlling the accuracy of determination of gaseous metabolic rates . This study showed the importance of controlling the temperature of gaseous oxygen entering the system . With such temperature control, it was possible to obtain data with an accuracy of +/- 5% at the 95% confidence level . Another source of error, the use of bi-carbonate buffer, was studied . A mathematical model was used to compensate for the affect of such buffers on the determination of carbon dioxide production rates . The use of the system for the continuous determination of gaseous metabolism during the growth and production phase for recombinant CHO cell cultures is described. Cytotechnology, 1994, 14(3), 157 - 65 Mass spectrometry: a tool for on-line monitoring of animal cell cultures; Behrendt U et al.; The magnetic sector mass spectrometer is able to detect oxygen uptake and carbon dioxide production rates from animal cell cultivations performed in 101 bioreactors . Such data have not been available with the use of classic exhaust gas analysis applying paramagnetic analyzers and infra-red sensors due to the insensitivity of the apparatus available . In the course of the present work we were able to demonstrate, that the oxygen uptake rate correlates to the number of viable cells . Additionally oxygen uptake rates supplied on-line information about the actual physiology of the cells: When the rates changed during the cultivation process, this immediately indicated the occurrence of limitations of components in the medium . The information could be useful in timing key events, such as performing splits or harvesting the bioreactor. Cytotechnology, 1994, 14(1), 67 - 80 Bead-to-bead transfer of Chinese hamster ovary cells using macroporous microcarriers; Ohlson S et al.; Chinese hamster ovary cells (CHO-K1) were cultivated in macroporous gelatin microcarriers (CultiSpher G and CultiSpher S) in spinner flasks and a 51 bioreactor . Near-to-confluent cultures were harvested by bead-to-bead transfer where intact microcarriers with cells were transferred from a spinner flask to another spinner flask or to the bioreactor with naked microcarrier beads . Successful bead-to-bead transfer was achieved in various split ratios . The duration of attachment seemed to be important where the direct contact of beads to each other can be achieved by intermittent stirring . Repeated transfers were performed and at least four transfers in spinner flasks were achieved . Two variations of bead-to-bead transfer were performed in the 51 bioreactor either by seeding the bioreactor with near-to-confluent beads cultivated in spinner flasks or in situ transfer by adding fresh beads to the bioreactor . As in the spinner case, attachment was achieved by intermittent stirring where donor beads were in close proximity to the acceptor beads . Again successful transfers were obtained as evidenced by the good growth on acceptor beads where cell yields were in the range of 3100-4500 cells/bead . The results suggest that bead-to-bead transfer of CHO-K1 cells can be easily performed and do provide an alternative route to applications where dissolution techniques may not offer an efficient solution. Cytotechnology, 1994, 14(1), 61 - 6 High-density culture of FM-3A cells using a bioreactor with an external tangential-flow filtration device; Kawahara H et al.; A novel bioreactor system developed for high-density cultures of suspended mammalian cells is described using a tangential-flow filtration device outside the culture vessel to separate viable cells from spent medium . The filtration device is based on thin porous microfiltration membranes with a pore size of 0.20-0.65 microns . Because cells have a diameter of about 10-20 microns, they cannot permeate these membranes with the spent medium . So, allowing a perfusion culture to be created using this system . In most membrane filtration systems, clogging of the membranes has made long-term operation difficult . In this system, however, high pressure is not applied directly to the membrane, thus minimizing clogging . Also, clogging of the membrane was prevented by washing the membrane surface once a day, and increasing the membrane surface area . With this system, FM-3A cells were cultured and maintained at a high density of 3.0 x 10(7) cells/ml for two weeks, and a continuous culture was supported for as long as 34 days. Cytotechnology, 1994, 14(1), 1 - 9 The Super-Spinner: a low cost animal cell culture bioreactor for the CO2 incubator; Heidemann R et al.; The production of small quantities of monoclonal antibodies and recombinant proteins was carried out using a new low cost production system, the Super Spinner . Into a 1 1 standard Duran flask a membrane stirrer equipped with a polypropylene hollow fiber membrane was installed to improve the oxygen supply by bubble-free aeration . The aeration was facilitated by using the CO2 conditioned incubator gas, which was pumped through the membrane stirrer via a small membrane pump . The maximal oxygen transfer rate (OTRmax) of the Super Spinner was detected . For this purpose one spinner flask was equipped with an oxygen electrode . The OTRmax was measured by the dynamic method . The ratio of membrane length to culture volume was adapted corresponding to the oxygen uptake rate of the cells according to the desired cell density . A balanced nutrient supply resulted in an optimal formation and yield of products. Biotechnol Prog, 1994 Jan-Feb, 10(1), 60 - 4 Continuous beta-galactosidase production in insect cells with a p10 gene based baculovirus vector in a two-stage bioreactor system; van Lier FL et al.; Continuous production of polyhedra or baculovirus-expressed proteins in insect cell cultures is limited to about 4 weeks . The decrease in production has been ascribed to the interference of defective deletion mutants with wild-type baculoviruses . The deleted genome sequences include the polyhedrin gene (or the heterologous gene of interest); in the remaining part, the major late p10 gene is always maintained . In the present study, the productivity of a recombinant baculovirus with the lacZ gene from Escherichia coli cloned downstream of the p10 promoter at the p10 locus was investigated . It was hypothesized that this p10 promoter driven gene is preserved over a longer period of time in a continuously operated two-stage bioreactor system than foreign genes behind the polyhedrin promoter at the polyhedrin locus . In two separate runs, beta-galactosidase production with the p10-lacZ recombinant reached quasi-steady-state levels of 30 and 60 units/cm3 . Polyhedron production was about 3 x 10(6) and 6 x 10(6) polyhedra/cm3, respectively . However, both polyhedron and beta-galactosidase production decreased after about 30 days of relatively constant production . In the infection reactor, deletion mutants of the virus, which contained both the polyhedrin and lacZ gene, were predominant . Therefore, the presence of the polyhedrin or p10 gene alone in deletion mutants is not sufficient for prolonged expression; other genes involved in major late gene expression and not present in the deleted virus are probably necessary. Biotechnology (N Y), 1994 Jan, 12(1), 75 - 8 Measurements of the growth and distribution of mammalian cells in a hollow-fiber bioreactor using nuclear magnetic resonance imaging; Callies R et al.; We have used diffusion-weighted 1H NMR micro-imaging and localized spectroscopy techniques to monitor the growth and distribution of mammalian cells in a hollow-fiber bioreactor . Non-invasive NMR measurements of this type should also allow investigation of metabolic heterogeneity and assist in future designs of hollow-fiber systems. Hum Antibodies Hybridomas, 1994, 5(3-4), 98 - 104 Antibody production of a human EBV-transformed B cell line and its heterohybridoma and trioma cell line descendants in different culture systems; Gustafsson B et al.; Cells of an EBV-transformed human lymphoblastoid B cell line, producing antibodies directed against tetanus toxin, were fused with mouse myeloma cells (SP2/0) and with mouse-human heteromyeloma cells (SPAM-8) resulting in the formation of heterohybridoma and trioma cells, respectively . Antibody production of the three cell lines were studied under different culture conditions . All three cell lines produced antibodies in concentrations ranging from 2.6 to 6.4 micrograms ml-1 in spent medium from stationary flask cultures . Dialysis cultures of trioma and heterohybridoma cells resulted in concentrations of 36 and 20 micrograms ml-1, respectively, whereas no significant increase was obtained with the EBV-transformed cells . Trioma cells, cultured in a hollow fiber cartridge bioreactor produced antibodies in concentrations of average of 303 micrograms ml-1, whereas the EBV-transformed cells did not adapt to this system . Furthermore, trioma and heterohybridoma cells injected into the intraperitoneal cavity of SCID-mice, produced antibodies in ascites fluid in concentrations of 500 and 640 micrograms ml-1 respectively. Hum Antibodies Hybridomas, 1994, 5(3-4), 143 - 51 Galactosylation of human IgG monoclonal anti-D produced by EBV-transformed B-lymphoblastoid cell lines is dependent on culture method and affects Fc receptor-mediated functional activity; Kumpel BM et al.; Human monoclonal antibodies to the Rh D blood group antigen were produced by EBV-transformed B cell lines grown in serum-free medium in low density (LD) static cultures or high density (HD) hollow fiber bioreactors . Glycosylation analysis of the purified IgG was determined by the binding of anti-GlcNAc monoclonal antibody (GN7) and by analysis of oligosaccharides released by hydrazinolysis . The LD MAbs had only trace levels of agalactosyl oligosaccharides (G0), the major species (> 70%) being digalactosyl structures (G2) . The HD MAbs, by contrast, contained about 10% G0 and relatively high levels (over 50%) of monogalactosyl (G1) oligosaccharides . beta 1-4 galactosyltransferase activity in the LD cell lines was similar to that found previously for other EBV-transformed B cell lines . The predominant oligosaccharides of an IgG3 anti-D, BRAD-3, contained bisecting N-acetylglucosamine . In functional assays with Fc receptor (Fc gamma R) positive effector cells, the highly galactosylated LD form of BRAD-3 was more active than the HD form in monocyte (Fc gamma RI) and K cell (Fc gamma RIII) mediated lysis of erythrocytes in ADCC assays, although these preparations showed no difference in Fc gamma RI-mediated rosette formation with U937 cells . One MAb, JAC10, was over 10-fold less active than two other IgG1 MAbs, 2B6 and BRAD-5, at mediating lysis of erythrocytes by Fc gamma RIII+ K cells; differences in sialylation may have contributed to this heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS) Adv Exp Med Biol, 1994, 368, 165 - 71 Use of hepatocyte cultures for liver support bioreactors; Gerlach JC; Hybrid artificial liver systems are being developed as extracorporeal temporary liver support therapy . Here, an overview is given with emphasis on hepatocyte culture models for bioreactors, in vitro studies, animal studies and the clinical application of hybrid liver support systems . In vitro studies show long term external metabolic functions of primary isolated hepatocytes in bioreactors . These systems are capable of supporting essential liver functions . Animal experiments show the possibility of upscaling the bioreactors for clinical treatment . Since there is no reliable animal model for investigations on the treatment of acute liver failure, the promising results of these studies have limited relevance . The small number of clinical studies are not sufficient to give statements about a clinical improvement of therapy of acute liver failure . Although important progress has been made in the development of the systems, multiple different hepatocyte culture models and bioreactor constructions are discussed in the literature, indicating competition in this field of medical research. Blood Cells, 1994, 20(2-3), 482 - 90; discussion 491 Expansion in bioreactors of human progenitor populations from cord blood and mobilized peripheral blood; Van Zant G et al.; Umbilical cord blood (UCB) and mobilized peripheral blood (MPB) provide an alternate source to bone marrow for transplantation . Expansion in vitro of stem/progenitor cell populations from these sources may provide adult-sized grafts otherwise not attainable because of the limited cell numbers available in the case of UCB or because of numerous rounds of apheresis required for sufficient MPB cells . We asked whether continuous perfusion culture could be employed in ex vivo expansion to produce clinically relevant numbers of stem/progenitor cells from these sources . To evaluate MPB, 1-10 million leukocytes, from patients who had received either granulocyte colony-stimulating factor (G-CSF) or cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF), were inoculated into bioreactors, with or without irradiated, allogeneic stroma . The growth factor combination in the perfusion medium consisted of interleukin-3 (IL-3), stem cell factor (SCF), GM-CSF and erythropoietin (Epo) . Under the best conditions tested, total cell numbers, granulocyte-macrophage colony-forming units (CFU-GM), and long-term culture-initiating cell (LTC-IC) populations were expanded by about 50-, 80-, and 20-fold, respectively, over 14 days . At low cell inocula (1 million), the presence of stroma enhanced the expansion of total cells and CFU-GM but not of LTC-IC . When SCF was not included in the medium, both total cells and CFU-GM expanded to a much lesser extent, but again the expansion of LTC-IC was not affected . At the higher cell inoculum (10 million), expansions of total cells and CFU-GM were equivalent with or without stroma . To evaluate UCB, cells were placed into bioreactors with or without irradiated, allogeneic stroma, and the bioreactors were perfused with medium containing the four standard growth factors . After 6-14 days, in several independent experiments, 20-24 million cells were harvested from bioreactors perfused with SCF-containing medium, irrespective of the presence or absence of preformed stroma . Similarly, in reactors perfused with SCF-containing medium (with or without stroma), an average 40- to 60-fold expansion of CFU-GM was obtained, yielding an average of 1.5-1.8 x 10(5) CFU-GM per reactor . Harvested cells were thus up to 40-fold enriched in CFU-GM in comparison to the inoculum . In the absence of SCF, cell expansions averaged 1.5- to 2-fold, and CFU-GM were expanded only 10- to 14-fold by day 14 . As before, the presence of preformed stroma did not affect either cell or CFU-GM yields, provided the cell inoculum was at least 4.5 million cells.(ABSTRACT TRUNCATED AT 400 WORDS) Eur J Cancer, 1994, 30A Suppl 3, S2 - 6 Applications of recombinant DNA technology in the production of glycosylated recombinant human granulocyte colony stimulating factor; Holloway CJ; Lenograstim has been developed by recombinant DNA technology and is expressed in large-scale mammalian cell culture . It has been shown that lenograstim is indistinguishable in its physicochemical, structural and biological properties with respect to native granulocyte colony stimulating factor isolated from a human cell line . In particular, both the recombinant and natural proteins have identical amino acid sequences, contain the same intra-polypeptide chain disulphide bridges and exhibit the same posttranslational carbohydrate structures . Lenograstim is manufactured by expanding inoculum from vials of the Manufacturer's Working Cell Bank (from molecular cloning) followed by culture in a large bioreactor . Purification of lenograstim involves a four-step chromatographic process . The active ingredient is monitored by in-process controls at all stages of manufacture and routinely as purified bulk . The finished product is formulated into excipients reflecting conditions close to the natural environment of the protein with respect to pH, osmolarity and the presence of human serum albumin. Dev Biol Stand, 1994, 83, 55 - 64 Hybridoma stability; Castillo FJ et al.; Hybridoma stability issues include mutations, chromosome losses, and the potential effects of process variables on the yield, quality and homogeneity of the Monoclonal Antibody (MAb) product . MAb production by murine hybridomas is typically unstable in the early stages after fusion but repeated cloning normally produces stable clones . The stability of hybridomas and the consistency of the MAbs produced during extended high density perfusion cultures at Xoma Corporation were evaluated . Cell stability was assessed by recovering cells from the bioreactors at different intervals and comparing their growth and product formation kinetics and yields to those of cells started fresh from the corresponding Manufacturer's Working Cell Banks . Product consistency was evaluated in the crude harvests and in the corresponding purified MAb lots by biochemical and functionality tests including: SDS-PAGE (reducing and non-reducing), IEF, HPLC (size exclusion and cation exchange), peptide mapping, N-terminal sequencing, carbohydrate composition and binding assays . Several murine hybridomas were studied during runs lasting several months and found to be stable by all criteria employed . Such results support the viability of extended hollow fiber perfusion cultures for reproducible production of murine MAbs . Selecting stable clones and understanding the effects of process variables on the quantity and quality of the MAbs are keys to controlling hybridoma stability during the manufacturing process. Gene, 1993 Dec 15, 135(1-2), 37 - 47 The origin of genetic information: viruses as models; Eigen M; A living entity can be described as a complex adaptive system which differs from any, however complex, chemical structure by its capability of functional self-organization based on the processing of information . If one asks, where does this information come from and what is its primary semantics, the answer is: information generates itself in feedback loops via replication and selection, the objective being 'to be or not to be' . This paper describes the theoretical framework of information-generating systems and provides experimental clues for some basic forms of genetic organization, such as molecular quasi-species, hypercyclic and compartmentalized RNA-protein assemblies . The results are primarily obtained with RNA viruses and virus-like systems . The experiments are carried out with the help of automated, computer-controlled bioreactors, called 'evolution machines', that may form the basis of a new 'evolutionary biotechnology'. Biotechnol Appl Biochem, 1993 Dec, 18 ( Pt 3), 217 - 26 Methanol detoxification by enzyme-loaded erythrocytes; Magnani M et al.; Alcohol oxidase (AlOx) from Pichea pastoris (a methylotrophic yeast) was encapsulated into human and murine erythrocytes up to 2 units/ml of packed cells . This enzyme has a much higher affinity for methanol than for ethanol, thus making the loaded erythrocytes useful cellular bioreactors able to catabolize methanol . Enzyme-loaded erythrocytes showed an increased rate of the hexose-monophosphate-shunt activity and a significant methaemoglobin production . However, the in vivo survival of these cells does not seem to be significantly affected by methanol catabolism . In vivo, mice receiving AlOx-loaded erythrocytes were able to keep the blood methanol concentrations below values that were about 50% of those found in mice receiving unloaded cells and similar amounts of methanol . Thus AlOx-loaded erythrocytes may add an important contribution to the detoxification protocol against methanol poisoning. Anal Chem, 1993 Dec 1, 65(23), 3363 - 7 Automated process monitoring of monoclonal antibody production; Paliwal SK et al.; Antifibronectin, monoclonal antibody was monitored through 52 h of production . Samples were automatically drawn from a bioreactor into the injection valve of an HPLC system without prior sample preparation . The hybridoma cell line was nonadherent, so whole cells were injected directly onto the perfusable protein A affinity column . There was only a modest column back pressure (ca . 1700 psi at a linear flow rate of 1.5 cm/s) after over 75 injections over the 52-h experiment . These experiments demonstrate the utility of high-speed chromatography for rapid process monitoring. J Immunol, 1993 Dec 1, 151(11), 5948 - 54 Reversion of the SCID phenotype by human T cell grafts . Development of cross-species immunocompetence; Donjon CM et al.; Due to defective recombinase function, mice with severe combined immunodeficiency (SCID) lack functional lymphocytes and can accept human lymphoid xenografts . Xenografted animals (SCIDhum) are thought to provide a neutral environment for in vivo studies of normal, malignant or HIV-infected human cells . SCIDhum often develop endogenous, EBV+ lymphomas in the graft and in the our study two-thirds of 142 SCIDhum mice did so . Surprisingly, one-third of animals developed reversion of the SCID phenotype rapidly after human T cell engraftment . 90% of tumors occurred in nonrevertant and only 10% in revertant mice . These revertant animals showed immunologic tolerance for normal human B lymphocytes, maintained stable levels of mouse and human IgM and IgG . In addition, they generated competent mouse T cells able to kill transformed (EBV+) but not fresh B cells from the same donor nor unrelated human B cell lines . The tolerance for human lymphoid cells and the cross-species antitumor competence of host T lymphocytes imply unexpected recognition and selection events . Rather than a neutral "bioreactor," these observations mark the SCID host as potentially active participant in a composite immune system generated by xenografting. Int J Artif Organs, 1993 Dec, 16(12), 843 - 6 Hepatocyte aggregate culture technique for bioreactors in hybrid liver support systems; Gerlach JC et al.; Utilizing a modified culture technique for hepatocytes, a high performance suspension culture is possible in which hepatocytes spontaneously form cell aggregates . The aggregates of 20-100 cells have been histologically confirmed to hold a three-dimensional structure, they show a long-term external metabolism and a survival time comparable with standard adhesion cultures . This technique has several advantages in the construction of large scale bioreactors for hybrid liver support systems. Braz J Med Biol Res, 1993 Dec, 26(12), 1305 - 17 Preparation of human rabies vaccine in VERO cell culture using a microcarrier system; Mendonca RZ et al.; 1 . The rabies virus (Pasteur PV strain) was propagated in VERO cells attached to microcarriers in a 3.7-1 bioreactor . Virus titers of about 10(6) LD50/ml were obtained regularly . 2 . Ultrafiltration was efficient for concentrating the virus suspensions, and the sucrose gradient reduced the residual VERO cell DNA to acceptable levels (less than 50 pg/dose) . The remaining cell DNA content was evaluated by dot-blot hybridization with a probe prepared with VERO cell DNA . 3 . The final virus preparations were inactivated by B-propiolactone treatment, showed a potency higher than 2.5 IU/dose and protected mice experimentally infected intracerebrally with rabies virus (CVS-13.2) . 4 . This methodology for the production of a rabies vaccine for human use should be of interest to countries where high technology facilities are not available. Analyst, 1993 Nov, 118(11), 1361 - 5 Simultaneous determination of ammonia nitrogen and L-glutamine in bioreactor media using flow injection; Palsson BO et al.; A novel split stream flow injection (FI) system suitable for the simultaneous determination of L-glutamine and ammonia nitrogen (ammonia-N) in cell culture media is described . Potentiometric detection of ammonia-N in one portion of the manifold is achieved using a commercial ammonia gas-sensing electrode fitted with a wall-jet cap . L-Glutamine is quantified in the other part of the split sample by potentiometric detection of ammonium ions (by an ammonium-selective polymer membrane electrode), liberated from the hydrolysis of glutamine after the sample flows through a glass bead reactor containing immobilized glutaminase . Endogenous ammonia-N and potassium ions that would normally interfere with the glutamine measurement are removed upstream using a unique tubular cation-exchange unit . Using 50 microliters sample volumes and mixed solutions of ammonium chloride and L-glutamine in Iscove's Modified Dulbecco's Medium to calibrate the FI measuring system, values for ammonia-N and L-glutamine determined for 22 media samples obtained from a bioreactor growing retroviral producer cells correlate well with those measured with commercial, manual enzymic-spectrophotometric assay kits. Am J Surg, 1993 Nov, 166(5), 512 - 21 Evolution of the bioartificial liver: the need for randomized clinical trials; Nyberg SL et al.; The pursuit of a bioartificial liver is well documented in the literature . Early techniques of artificial liver support that have undergone clinical testing included simple exchange transfusions, extracorporeal xenogeneic or allogeneic liver perfusion, cross-circulation, hemodialysis, charcoal hemoperfusion, and plasmapheresis with plasma exchange . These techniques failed because they were unable to adequately support those hepatic functions essential for survival and because they lacked a back-up therapy, such as liver transplantation, for irreversible forms of liver disease . The concept evolved that hepatic functions essential for survival would be best performed by hepatocytes in an apparatus that allowed sustained or repetitive application . The best results have been achieved with bioartificial liver technologies that employ hepatocytes as implantable systems or extracorporeal devices . Implantable bioartificial liver systems include hepatocytes that have been on coated microcarrier beads, within microencapsulated gel droplets, within biodegradable polymeric substrates, or as spheroid hepatocyte aggregates . Extracorporeal systems include hepatocytes in suspension, on flat plates, and in hollow fiber bioreactors . Several extracorporeal systems have undergone extensive animal testing and are entering the early stages of human clinical trials . Randomized trials are needed to establish the value of bioartificial liver support in the treatment of patients with acute hepatic failure or as a bridge to liver transplantation. Cell Transplant, 1993 Nov-Dec, 2(6), 453 - 60 Correction of bilirubin conjugation in the Gunn rat using hepatocytes immobilized in alginate gel beads as an extracorporeal bioartificial liver; Fremond B et al.; A new extracorporeal bioartificial liver using alginate-entrapped hepatocytes was developed and evaluated for its ability to correct the lack of bilirubin conjugation in the Gunn rat . Hepatocytes were harvested from Sprague-Dawley rats by the two-step collagenase perfusion method and then immobilized in Ca(++)-alginate beads . The ability of immobilized hepatocytes to conjugate bilirubin was investigated in vitro by comparison with hepatocyte monolayer cultures . The bioartificial liver consisted of a cylindric bioreactor containing either alginate beads with hepatocytes (test group) or alginate beads alone (control group) . Gunn rats were connected to this bioreactor via an extracorporeal circulation and bile fractions were collected at hourly intervals . Both bilirubin monoconjugates and bilirubin diconjugates were measured in the bile by high pressure liquid chromatography . Hepatocyte viability in alginate beads was determined prior to and at the end of each experiment and found to be unchanged (75%) . In the test group, the concentration of bilirubin conjugates increase rapidly, attaining median values of 72.26 microM and 92.59 microM for mono and diconjugated bilirubin respectively, during a 3 h period of extracorporeal circulation . In the control group, the levels of either conjugate did not exceed 0.87 microM throughout the experiments . Statistical analysis showed a significant difference between the two groups (p < 0.0023) . These results suggest that the bioartificial liver used in this study represents an effective method for the temporary correction of the Gunn rat's genetic defect . Such a system might be of therapeutic interest in acute liver failure. Appl Microbiol Biotechnol, 1993 Nov, 40(2-3), 246 - 50 Immobilization of the slime mould Dictyostelium discoideum for the continuous production of recombinant human antithrombin III; Tiltscher H et al.; Recombinant vegetative Dictyostelium discoideum cells were immobilized inside a porous matrix by an inorganic membrane that was permeable to nutrients but not to cells, in order to produce recombinant human antithrombin III . Cells so entrapped could reach up to 15 times higher biomass densities compared with organisms growing freely in suspension . The high cell concentration maintained in the immobilized cell bioreactor caused an increase in specific and volumetric productivity . In continuous operation a maximum volumetric antithrombin productivity of 56 ng h-1 ml-1 catalyst bulk volume was attained at a dilution rate of 0.016 h-1 . In addition, the good retention of metabolic activity for several weeks as well as the convenient form of storage and regeneration of the catalytic system were shown. Biotechnol Prog, 1993 Nov-Dec, 9(6), 675 - 8 Production of human alkaline phosphatase, a secreted, glycosylated protein, from a baculovirus expression system and the attachment-dependent cell line Trichoplusia ni BTI-Tn 5B1-4 using a split-flow, air-lift bioreactor; Chung IS et al.; A split-flow, air-lift bioreactor for the cultivation of insect cells to produce recombinant protein is described . It can be used advantageously with attached cell systems . This bioreactor incorporates two sections: a rise and a downcomer . Trichoplusia ni BTI-Tn 5B1-4 cells are grown on a support material of glass beads or microcarriers placed in the downcomer . This cell line is more productive than other commonly used insect cell lines, but it has the disadvantage of being difficult to use at large volumes since it is not easily adaptable to suspension culture . Adequate oxygen demand is supplied by sparging without direct exposure of cells to air bubbles . Nutrients are supplied convectively to the attached cells on support material as the fluid flows through the downcomer . The split-flow, air-lift bioreactor appears to be suitable for insect cell culture and is potentially scalable . It can provide a high surface-to-volume ratio and can be operated in batch or continuous mode . A lab-scale prototype bioreactor has been constructed and tested for the production of a secreted, glycosylated recombinant protein (human alkaline phosphatase) or seAP using an Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) vector . With a ratio of riser cross-sectional area to downcomer cross-sectional area of 1, an aspect ratio of 4.4, an air-flow rate of 54 mL/min in the riser, and a bed of 2400 3-min nonporous glass beads, 10.7 micrograms/mL of seAP was produced using an MOI (multiplicity of infection or the ratio of plaque-forming units to cells) of 10.(ABSTRACT TRUNCATED AT 250 WORDS) Biotechnol Prog, 1993 Nov-Dec, 9(6), 666 - 70 In situ fluorescence cell mass measurements of Saccharomyces cerevisiae using cellular tryptophan; Horvath JJ et al.; This work describes a new spectroscopic optical fiber/rod technique for in situ real time measurement of cell mass and product concentrations in bioreactors using intrinsic fluorescence . The variable excitation/emission wavelength capability of this sensor allows for species-selective measurement during fermentations . Cell mass (tryptophan) and product concentrations (pyridoxine) have been measured during fermentations of Saccharomyces cerevisiae . The effects of varying substrate concentration and oxygen concentration on the observed cell mass signals are eliminated by direct measurement of cell mass, as opposed to indirect measurement schemes such as those using NADH fluorescence . The sensor is robust and able to undergo many cycles of in situ steam sterilization without degradation, and its fluorescence signal is linear with concentration for all species studied in this work . Tryptophan fluorescence from yeast is shown to be a better measure of cell mass than NADH fluorescence. Biotechnol Prog, 1993 Nov-Dec, 9(6), 580 - 6 Development of scale-down techniques for investigation of recombinant Escherichia coli fermentations: acid metabolites in shake flasks and stirred bioreactors; Dahlgren ME et al.; We have developed shake-flask screening conditions that are predictive of specific expression of the chimeric toxin, TGF alpha-PE40, by recombinant Escherichia coli JM109 in stirred bioreactors . When a nutrient-rich stirred bioreactor medium was used in shake flasks, neither the extent of growth nor the specific level of recombinant protein expression duplicated the performance in stirred bioreactor fermentations . Incomplete oxidation of glucose and concomitant accumulation of organic acid metabolites, as well as oxygen limitation and lack of pH control, were examined as contributors to the poorer performance in the flask . The medium buffering capacity, initial glucose level, and flask aeration were evaluated to establish the limits of "scale-down" conditions for expression both in a complex nutrient medium (M101) similar to that used in stirred bioreactors and in a defined (FM) medium . Acid metabolites and ethanol were measured as indicators of carbon flow from glucose as well as indirect indicators of oxygen limitation . For the complex M101 medium, optimal shake-flask performance in 250-mL, nonbaffled flasks at 37 degrees C occurred with 0.3 x medium strength, supplementation with 0.3 m HEPES buffer (pH 7.5), and 10 mL of medium per flask . Cultures grown under these conditions produced a maximum density of 3.6 g of dry cell weight/L (as estimated by absorbance measurements at 600 nm) and maintained a pH near neutrality . Additionally, metabolite markers of anaerobic or microaerobic conditions, such as ethanol, lactate, and pyruvate, were not detected, and specific expression of TGF alpha-PE40 was comparable to stirred bioreactors induced for expression at various biomass levels.(ABSTRACT TRUNCATED AT 250 WORDS) J Biotechnol, 1993 Nov, 31(2), 205 - 17 Fed-batch culture of insect cells: a method to increase the yield of recombinant human nerve growth factor (rhNGF) in the baculovirus expression system; Nguyen B et al.; A fed-batch method was developed which increased the density of insect cells (Spodoptera frugiperda, Sf-9 cells) in suspension culture and the feeding of nutrients improved the yield of a recombinant protein produced by a baculovirus expression system . Analysis of spent medium samples indicated that depletions of glucose and glutamine correlated with the retardation of cell growth . Feeding of a mixture of nutrients consisting of glucose, glutamine, yeastolate and lipids solution restored the growth rate . In fed-batch culture, cell density was increased from 3 x 10(6) cells per ml to 1.2 x 10(7) cells per ml and the increased cell density enhanced the yield of the desired recombinant product, in this case, human nerve growth factor (rhNGF) . The optimal conditions for the production of rhNGF were also defined by selecting the appropriate viral multiplicity of infection (MOI) . At a cell density of 5 x 10(6) ml-1, a MOI of 0.05 (plaque forming units per cell) gave the highest yield of rhNGF in culture fluid 3 d post-infection . The yield of rhNGF was 20 mg l-1 . The fed-batch method was scaled up to 12 l stirred bioreactor. J Biotechnol, 1993 Nov, 31(2), 147 - 60 Search for cell proliferation markers suitable for cell count in continuous immobilized cell bioreactors; Capiaumont J et al.; The control of hybridoma cell cultures in bioreactors requires the use of convenient indicators to monitor the proliferation of the biomass . In order to select appropriate indications, we followed the variations of several compounds including tumoral markers, polyamines, sialic acids, purine and pyrimidine bases, enzymes and metabolites such as glucose, lactate and amino acids, and the variations of cell density during batch culture . Significant correlations were found between the number of viable cells and alkaline phosphatase, beta-glucuronidase, glucose and lactate measured in the culture medium of hybridoma strains . The correlation calculated from alkaline phosphatase and beta-glucuronidase concentrations in culture medium underestimated cell number . The correlation established with glucose and lactate gave the best indication of cell proliferation in continuous culture with an immobilized cell bioreactor . Finally, the exact quantification of the biomass in these culture conditions can be obtained using the mean of glucose and lactate correlations. Enzyme Microb Technol, 1993 Nov, 15(11), 928 - 35 Continuous enzymatic production of oligopeptides: synthesis of an enkephalin pentapeptide in a multistage bioreactor; Richards AO et al.; A feasibility study of the continuous enzymatic production of short oligopeptides was undertaken using the synthesis of {Leu5}-enkephalin pentapeptides as a model system . A three-stage bioreactor was designed to perform the independent syntheses of the constituent tri- and dipeptide fragments and their subsequent condensation . Both the N-terminal (N-X-L-Tyr.Gly.GlyOEt) and C-terminal (L-Phe.LeuNH2) peptides were prepared in 75-85% yield in column reactors packed with Celite-immobilized alpha-chymotrypsin . The enkephalin pentapeptide was obtained in up to 30% yield in the third bioreactor module containing Celite-immobilized proteinase K . The bioreactor was run continuously for over 1,000 h, producing, under steady-state conditions, up to 0.7 g day-1 of the pentapeptide. Am Biotechnol Lab . 1993 Nov;11(12):26. Application of a hollow fiber membrane cell culture system in medicine; Marx U et al.; The Tecnomouse system is useful for cultivating transformed cell lines producing MAbs or recombinant proteins, but human tumor cells can also be propagated for autologous immunization protocols or research properties . Lymphokine-activated killer cell production and stem cell proliferation seem to be possible . Moreover, primary human cells of lymphoid organs can be successfully kept viable over long periods of time in a three-dimensional, tissue-like culture . Therefore, the bioreactor is a tool for the in vitro modulation of a variety of human organs. Biotechnology (N Y), 1993 Nov, 11(11), 1263 - 70 Transgenic livestock as bioreactors: stable expression of human alpha-1-antitrypsin by a flock of sheep; Carver AS et al.; We have previously described the generation of transgenic sheep expressing human alpha 1-antitrypsin (h alpha 1AT) in their milk . Here, we report the fidelity of transgene transmission and expression by these animals and their progeny . Transgene transmission has been demonstrated in four of six ovine lines studied . Three of these four lines have exhibited stable transmission of the transgene, whereas the fourth has produced some offspring with reduced copy numbers . Sequential lactations of founder animals has yielded very similar levels of h alpha 1AT protein in milk . Moreover, in one line, derived from a founder male, a flock of seven G1 ewes have yielded comparable levels of h alpha 1AT protein in first and second lactation milk . Two G2 ewes of this line have also produced levels of human protein equivalent to their mother . Although the inheritance of the same transgene in mice was reminiscent of the situation in sheep, stable expression was observed in only one or four lines studied . The importance of these observations to the use of transgenic livestock as bioreactors for the production of human proteins is discussed. Endocrinology, 1993 Oct, 133(4), 1490 - 503 Purification and characterization of recombinant human thyrotropin (TSH) isoforms produced by Chinese hamster ovary cells: the role of sialylation and sulfation in TSH bioactivity; Szkudlinski MW et al.; The biological significance of glycosylation variants of pituitary glycoprotein hormones remains controversial because of the indirect methods usually employed to determine carbohydrate composition or structure as well as the use of unreliable biological/immunological ratio to determine bioactivity . We have previously characterized recombinant human TSH (rhTSH) secreted by Chinese hamster ovary cells attached to microcarrier beads in a large scale bioreactor after stable transfection of hCG alpha and hTSH beta minigenes . In the present study rhTSH has been used as a model to determine structure-function relationships of different isoforms of glycoprotein hormones . We have now produced greater than 200 mg rhTSH using a hollow fiber bioreactor . The highly purified rhTSH produced in the hollow fiber bioreactor (rhTSH-N) as well as rhTSH commercially produced in a large scale bioreactor (rhTSH-G) were quantitated by immunoassays, receptor binding assay, and amino acid analysis and further characterized by a variety of physico-biochemical methods, including chromatofocusing and carbohydrate analysis . rhTSH-G, rhTSH-N, as well as pituitary human TSH (phTSH) have been separated by chromatofocusing on a Mono P column into several isoforms with different pI values . Compositional analysis of the fractions showed higher sialic acid content in the more acidic rhTSH-G fractions . phTSH acidic isoforms showed higher total sulfate and sialic acid contents than the more basic fractions . The bioactivities of various TSH isoforms based on rigorous quantitation of mass by amino acid analysis determined in three different FRTL-5 cell bioassays showed that the more basic and less sialylated fractions of rhTSH-G were more active than the more acidic fractions . In contrast to the in vitro data, highly sialylated and acidic rhTSH-G isoforms showed longer plasma half-lives and higher in vivo bioactivity than the basic forms . These results indicate that secreted rhTSH, similar to intrapituitary phTSH, exists as a mixture of charge isoforms that are related at least in part to the degree of sialylation . The degree of sialylation, highly dependent on the bioreactor production conditions, appears to be the major factor affecting the charge heterogeneity, MCR, and bioactivity of rhTSH. J Pharm Biomed Anal, 1993 Oct, 11(10), 921 - 6 On-line monitoring of urea in effluent liquid during haemodialysis; Orellana A et al.; An analytical system specially built for on-line urea monitoring is reported . Measurements are carried out in the effluent of a haemodialysis machine . The measuring system employs the dialyser inflow stream as a carrier solution channel in a continuous fashion . The analyser periodically samples the outflow stream of the dialyser by means of an automatic injection valve . The analyser features a bioreactor consisting of immobilized urease and a gas-diffusion module . It is through this module that the urea is converted to ammonia gas which is transferred to another carrier channel, this transports the ammonium ion to a tubular, all-solid-state, ion-sensitive electrode . A timer controls the transport, injection, the measuring and the recording subsystems . The analyser has been used during actual haemodialysis sessions . Urea clearances were also measured in batch, using conventional spectrophotometric clinical equipment . The correlation between both methodologies was sufficient to confirm the usefulness of the developed on-line analyser to monitor the optimal length of haemodialysis sessions. Arzneimittelforschung, 1993 Oct, 43(10), 1134 - 9 Mammalian cell cultures . Part I: Characterization, morphology and metabolism; Werner RG et al.; Primary cell cultures are obtained by trypsinization from tissue cultures usually as a monolayer culture . The absence of fetal calf serum will support suspended growth behaviour of spontaneously transformed cells . After several passages the cell line becomes more stable and gives rise to a continuous cell line . Such continuously growing cell lines are a prerequisite for production of recombinant DNA derived proteins . Mammalian cells are 10-100 times larger in diameter than microorganisms . They have no cell wall and express therefore a higher sensitivity to hydrodynamic sheer forces . One of the most stringent problems in mammalian cell culture are "silent" contaminants with mycoplasma which might change cell growth . Mammalian cell cultures show a complex metabolism where regulation of metabolites and catabolites are not fully understood . Glucose is the main carbohydrate source . Also three groups of intercorrelated amino acids are known . Lactate as the primary metabolite of glucose and ammonia as a metabolite of glutamine are expected to be cytotoxic for mammalian cells . Although in some experiments even the addition of ammonia has no significant effect on the viability of hybridoma cells . Adherent cells can be cultivated attached to surfaces such as microcarrier or wire springs . Suspended cells are grown in stirred bioreactors with a comparable technology to fermentation of microorganism . Parameters such as pH, temperature, stirring tip speed and osmolality have to be well controlled in order to obtain high cell viability and cell density. Biotechniques, 1993 Oct, 15(4), 674 - 83 Pilot-scale production of murine monoclonal antibodies in agitated, ceramic-matrix or hollow-fiber cell culture systems; Kurkela R et al.; The purpose of this research was to compare three bioreactor systems for the pilot-scale production of monoclonal antibodies (MAbs) . We needed to produce gram quantities of murine MAbs against human prostatic acid phosphatase for use in fragmentation, radiolabeling and in vivo radio-imaging studies . The stable hybridoma cell line secreting IgG1 antibodies was chosen for production . Of the available bioreactor systems, we chose to test an agitating 30-liter bioreactor in repeated batch mode, a ceramic-matrix bioreactor in both repeated batch and continuous perfusion modes and a hollow-fiber bioreactor in continuous perfusion mode . The highest cultured MAb yield, 151 +/- 126 mg/day (mean +/- SD, n = 22), was achieved in the 30-liter bioreactor in repeated batch mode with the MAb being harvested in a large volume of medium, giving a reactor productivity of 4.3 +/ 3.4 mg/liter/day (mean +/- SD, n = 22) . The most concentrated MAb was harvested from the continuously perfused hollow-fiber bioreactor, which had the highest reactor productivity, 307 +/ 142 mg/liter/day (mean +/- SD, n = 47) and an average rate of MAb production of 55.3 +/- 25.7 mg/day (mean +/- SD, n = 47) . Taking the use of serum into consideration, the cost of MAb production was lowest in the continuously fed and harvested ceramic-matrix and hollow-fiber cell culture systems . A compact blood glucose meter proved to be a novel and suitable device for the rapid monitoring of glucose concentrations in hybridoma cultures. Trends Biotechnol, 1993 Oct, 11(10), 413 - 8 Biocatalysis in the gas phase; Lamare S et al.; The biocatalysis of substrates in the gas phase may offer advantages over many conventional solution-based reactions, both in analytical devices and in bioreactors designed to accommodate this new technology . To date, however, the range of substrates for which gas-phase biocatalysis has been shown to be suitable is limited . Further research is required to establish the parameters that affect the kinetics and productivity of such systems. Bioconjug Chem, 1993 Sep-Oct, 4(5), 341 - 6 Temperature-responsive bioconjugates . 2 . Molecular design for temperature-modulated bioseparations; Takei YG et al.; We have synthesized carboxyl semitelechelic oligo(N-isopropylacrylamide) (OIPAAm) using radical telomerization with 3-mercaptopropionic acid . This telomerization is also effective for the synthesis of carboxyl semitelechelic co-oligomers of IPAAm with butyl methacrylate (BMA) as hydrophobic or N,N-dimethylacrylamide (DMAAm) as hydrophilic comonomers . All co-oligomers are highly water-soluble at lower temperatures and exhibit phase separation with increasing temperature . Pure OIPAAm exhibits a lower critical solution temperature (LCST) at 32 degrees C, and the LCST for co-oligomers can be controlled to increase over 32 degrees C with increasing DMAAm composition and to decrease below 32 degrees C with increasing BMA composition . OIPAAm was grafted to bovine serum albumin (BSA) and bovine plasma fibrinogen (BPF) by activated ester-amine coupling . These OIPAAm-biomolecule conjugates maintain their temperature responses, are soluble in cold water, and precipitate over a range of temperatures related to oligomer content . Conjugates could be selectively precipitated and independently separated from conjugate solution mixtures with increasing temperature . In this case, the number of OIPAAm molecules attached to a conjugate affects the aggregate sizes of precipitated conjugates in mixtures . Both conjugate mixture ratios and solution concentrations influence the contamination of oligo(IPAAm-co-DMAAm)-BSA conjugates in precipitated oligo(IPAAm-co-BMA)-BPF conjugates . Furthermore, precipitated conjugates separated using centrifugation and filtration redissolve in water and maintain their biofunctionality, indicating the potential of strategy in reversible bioreactors and protein separations. J Biochem Biophys Methods, 1993 Sep, 27(2), 105 - 16 A new NMR airlift bioreactor used in 31P-NMR studies of itaconic acid producing Aspergillus terreus; Lyngstad M et al.; An airlift bioreactor for in-vivo NMR studies of cells is described . The 10-mm diameter airlift reactor was constructed for studies of mycelial/pellet forming organisms, grown in suspension . With this device 161 MHz 31P-NMR spectra of living Aspergillus terreus cells, producing itaconic acid, have been obtained . Signals were observed for intra- and extracellular orthophosphate, glycerol-3-phosphorylethanolamine (GPE), glycerol-3-phosphorylcholine (GPC), sugar phosphates and polyphosphate . The spectra also showed broad overlapping resonances in the shift range of NAD(H) and NADP(H) . Polyphosphate disappeared when the respiratory gas was exchanged for pure N2 . The intracellular pH was estimated at 6.2 . In spectra of cell extracts approx . 60 peaks were observed i