Curr Opin Biotechnol, 1998 Apr, 9(2), 152 - 6 Apoptosis in cell culture; al-Rubeai M et al.; Most cells can exhibit a biochemical pathway which mediates their own destruction in a highly controlled and genetically defined manner . In animal cells, a morphologically distinct form of this 'programmed cell death' has been identified and extensively characterised . This phenomenon, which has been named apoptosis, accounts for most of the cell deaths that take place during the production of biopharmaceuticals from animal cell lines . In the past few years, the factors responsible for the induction of apoptosis in the bioreactor environment have been identified . Furthermore, a growing number of studies have demonstrated that the suppression of apoptosis by the overexpression of anti-apoptosis genes, most notably bcl-2, result in improved culture productivity. Biol Pharm Bull, 1998 Apr, 21(4), 330 - 4 Non-antigenic and low allergic gelatin produced by specific digestion with an enzyme-coupled matrix; Sakai Y et al.; Porcine gelatin (heat-denatured collagen) was digested with a bioreactor using an enzyme-coupled matrix (ECM) with purified collagenase . The digested gelatin, FreAlagin type R (M.W . range 200-10000 Da), was further purified by an HPLC system depending upon molecular size . The molecular weight range of the purified fractions, FreAlagin type P and type AD, were 200-500 and 2000-10000 Da, respectively, and glycine was the N-terminal amino acid of both types (> or =93%) . ECM has the capability of digesting gelatin at a specific point in the sequence before glycine, and it was determined that FreAlagin type P consists of a tri-peptide fraction with the amino acid sequence Gly-X-Y . No types of FreAlagin exhibited any reactivity with gelatin-specific IgG antibody raised in guinea pigs, and they also possessed an extremely low reactivity with gelatin-specific IgE antibody from the sera of patients who had experienced an anaphylactic reaction against gelatin after vaccination or after eating gelatin-containing foods . From these results, it was determined that FreAlagin types R and AD were non-antigenic, low-allergic gelatins . FreAlagin type R, and especially type AD, had strong adsorption-blocking activity comparable to the level of bovine serum albumin, whereas type P and glycine had virtually no adsorption-blocking activity . Therefore, the new types of gelatin, FreAlagin types R and AD, are suitable for pharmaceutical use to avoid gelatin allergy. Protein Expr Purif, 1998 Apr, 12(3), 390 - 8 Expression, purification, and bioassay of human stanniocalcin from baculovirus-infected insect cells and recombinant CHO cells; Zhang J et al.; Stanniocalcin is a calcium- and phosphate-regulating glycoprotein hormone that was first described in fish where it functions in preventing hypercalcemia . Human cDNA clones encoding the homolog of stanniocalcin have been recently isolated . In this study, the full-length cDNA coding for human stanniocalcin (hSTC) was cloned into both baculovirus and CHO expression vectors . Recombinant hSTC was then produced efficiently from both baculovirus-infected insect cells and CHO cells in large-scale bioreactors . Purification protocols were developed and used to purify recombinant hSTC from both sources in four chromatography steps . The hSTCs from both expression systems were secreted as glycosylated proteins and as disulfide-linked homodimers . The results from glycosylation studies indicated that stanniocalcin from both sources contained N-linked oligosaccharides but no O-linked sugars . In an in vivo bioassay based on the inhibition of gill calcium transport in fishes, the baculovirus and CHO-expressed protein showed biological activity which is dose dependent . The inhibitory effects of hSTC produced from both systems were essentially equipotent in fishes, despite the differences in glycosylation . Consequently, the precise role of the carbohydrate moiety in recombinant hSTC remains to be determined . Protein Expr Purif, 1998 Apr, 12(3), 323 - 30 Baculovirus-mediated large-scale expression and purification of a polyhistidine-tagged rubella virus capsid protein; Schmidt M et al.; The capsid protein of rubella virus was produced in baculovirus-infected Spodoptera frugiperda insect cells, with a polyhistidine affinity tag at the carboxy terminus . The RV capsid recombinant protein was produced in a 10-liter bioreactor and purified, under nondenaturing conditions, using immobilized metal-ion affinity chromatography . Immunoblot analyses indicated that the purified recombinant protein was intact and migrated with the expected molecular weight . The final yield was 5 mg of purified protein per liter of cell culture . Surface plasmon resonance was used to investigate the antigenic potential of the histidine tagged capsid protein in an antigen-antibody interaction study . A specific interaction between the two proteins was shown . Our results suggest that this strategy should be useful in interaction studies of other virus-specific proteins and antibodies . J Leukoc Biol, 1998 May, 63(5), 550 - 62 Suppressed PHA activation of T lymphocytes in simulated microgravity is restored by direct activation of protein kinase C; Cooper D et al.; Utilizing clinostatic rotating wall vessel (RWV) bioreactors that simulate aspects of microgravity, we found phytohemagglutinin (PHA) responsiveness to be almost completely diminished . Activation marker expression was significantly reduced in RWV cultures . Furthermore, cytokine secretion profiles suggested that monocytes are not as adversely affected by simulated microgravity as T cells . Reduced cell-cell and cell-substratum interactions may play a role in the loss of PHA responsiveness because placing peripheral blood mononuclear cells (PBMC) within small collagen beads did partially restore PHA responsiveness . However, activation of purified T cells with cross-linked CD2/CD28 and CD3/CD28 antibody pairs was completely suppressed in the RWV, suggesting a defect in signal transduction . Activation of purified T cells with PMA and ionomycin was unaffected by RWV culture . Furthermore, sub-mitogenic doses of PMA alone but not ionomycin alone restored PHA responsiveness of PBMC in RWV culture . Thus our data indicate that during polyclonal activation the signaling pathways upstream of PKC activation are sensitive to simulated microgravity. Transfus Clin Biol, 1998 Feb, 5(1), 80 - 7 {Hepatocytes in cell therapy}; Clement B et al.; Cell-based therapy could represent an alternative treatment to orthotopic liver transplantation in acute liver failures and for the correction of genetic defects of various enzymatic functions . Several recent studies indicate that hepatocytes injected either in the spleen or in portal vein can restore liver-specific function(s) in animal model systems . Alternatively, an extracorporal hybrid bioartificial liver might provide liver-specific functions, maintain the patient alive and allow spontaneous recovery of the patient's own liver, or act as a bridge toward liver transplantation in acute liver failures . Various drawbacks of devices such as flat culture substrates, hollow-fiber bioreactors or microcarriers led us to develop a reliable extracorporeal bioartificial liver based on alginate-entrapped hepatocytes . This system was used successfully for the correction of the Gunn rat genetic defect which results in the lack of bilirubin conjugation . The development of this system for clinical purposes requires large yields of functional hepatocytes . We isolated porcine hepatocytes by collagenase perfusion of the liver and cells were immobilized within alginate beads which were subsequently inoculated in a bioreactor . Porcine hepatocytes expressed liver-functions at high levels, particularly those involved in detoxification and biotransformation processes; they were immunoisolated from immunoglobins and could be cryopreserved . This system represents a promising tool for the design of an extracorporeal bioartificial liver in human beings. Biotechnol Prog, 1998 Mar, 14(2), 265 - 9 Enhancing xanthan fermentations by different modes of glucose feeding Amanullah A, Satti S, Nienow AW. This paper is the fourth in a series aimed at improving the understanding and operation of conventional agitated fermenters for the production of the commercially important gum, Xanthan . In the first, reproducible fermentations were established and this protocol was used in studies of different agitator types and of bulk mixing and dissolved oxygen concentration in the next two . Here, building on the previous work, the influence of different glucose feeding strategies on Xanthan production in a 20-L agitated fermenter under equivalent conditions of agitation and dissolved oxygen is reported . The biological performances in three types of fed-batch cultures (a two-step glucose addition, multiple glucose-pulse feeding and continuous feeding of glucose) are compared to two batch fermentations with different initial glucose concentrations . The work confirmed that improved performance cannot be achieved by increasing the initial glucose concentration above 50 g/L nor by a single 10 g/L pulse addition (initial glucose concentration of 40 g/L) while significant nitrogen is still present . On the other hand, the simple pulse and continuous feeding strategies, after nitrogen has been essentially exhausted and under conditions of nonlimiting dissolved oxygen and similar bulk mixing, can result in a greatly enhanced performance compared to batch fermentations . Using the final Xanthan gum concentration, the yield on glucose and the overall productivity as performance indices, values of 62 g/L, 0.82 g of Xanthan/g of glucose, and 0.72 g/(L.h), respectively, were obtained compared to literature values for conventional stirred bioreactors of 15-30 g/L, 0.27-0.86 g of Xanthan/g of glucose, and 0 . 12-0.43 g/(L.h). Biotechnol Prog, 1998 Mar, 14(2), 203 - 9 Screening tool for hollow-fiber bioreactor process development Gramer MJ, Poeschl DM. Fundamental research of factors affecting cell growth in hollow-fiber bioreactors is hindered by the lack of an efficient screening tool . To address this issue, a hollow-fiber micro-bioreactor has been developed . Hollow fibers with 10 kDa molecular weight cutoffs are housed within a piece of silicone tubing . Cells are inoculated within the hollow fibers which provides a 0.2-mL culture volume . The space between the fibers and silicone tubing (5 or 16 mL) is used as a medium reservoir sufficient to feed the cells for at least 24 h . Oxygenation is provided directly through the silicone tubing so that a pump for medium recirculation is not required . As a result, many conditions can be tested simultaneously in a single incubator . Three days after inoculation at 5 x 10(6) cells/mL in the micro-bioreactor, the rho 1D4 murine hybridoma cell line reached 2.8 x 10(7) cells/mL with an antibody concentration of 0.17 mg/mL . When inoculated at 5 x 10(7) cells/mL, the cell concentration reached 1.8 x 10(8)/mL after 3 days with an antibody concentration of 1.0 mg/mL . Results from a series of experiments with the micro-bioreactor suggested that the initial growth phase of this cell line in a hollow-fiber system is dependent on the serum concentration in the medium reservoir . This prediction was tested by simultaneously inoculating two production-scale hollow-fiber bioreactor systems . The cell side of the membrane for each bioreactor contained 10% serum, but serum was added to the reservoir side of only one of the bioreactors . The cells with only basal medium in the reservoir died after a few days, while the cells with 10% serum in the medium reservoir grew rapidly . These results demonstrate that the micro-bioreactor developed here can support good cell growth and that it can be used as a research tool to predict the performance of large-scale hollow-fiber systems. J Biotechnol, 1998 Feb 5, 60(1-2), 47 - 54 Recombinant gene expression in Escherichia coli cultivation using lactose as inducer; Gombert AK et al.; The use of lactose as inducer of foreign gene expression under control of the lac UV5 promoter was investigated in recombinant Escherichia coli . Chicken muscle troponin C (TnC) gene was transcripted by T7 RNA polymerase and expressed in bioreactor cultivations after a feed-forward controlled fed-batch growth phase . Cell concentrations of 22 g l-1 dry cell weight (DCW)--before induction started--were used to achieve a TnC content of 19.5% of total cell protein through an induction strategy that combined the addition of a specific lactose amount of 4.7 g g-1 DCW divided into three pulses and the addition of yeast extract (1 g l-1) together with the second and the third lactose pulses . The results presented suggest that the residual lactose concentration plays an important role on the production of the heterologous protein. Exp Cell Res, 1998 Apr 10, 240(1), 58 - 65 Chondrogenesis in a cell-polymer-bioreactor system; Freed LE et al.; Chondrogenesis was studied under controlled in vitro conditions using a cell-polymer-bioreactor system . Bovine calf articular chondrocytes were seeded onto biodegradable polymer scaffolds and cultured in rotating bioreactor vessels . Concomitant increases in the amounts of glycosaminoglycan (GAG) and type II collagen resulted in cell-polymer constructs with continuous cartilaginous matrix over their entire cross sections (6.7 mm diameter x 5 mm thick) after 40 days of cultivation . As compared to natural calf cartilage, constructs had comparable cellularities, 68% as much GAG and 33% as much type II collagen per gram wet weight . The progression of chondrogenesis in chondrocyte-polymer constructs was similar to that suggested previously for precursor cells in vitro and developing limbs in vivo . In particular, the polymer scaffold provided a three-dimensional structure that could be seeded with chondrocytes at high cell densities in order to establish cell-to-cell contacts and initiate cartilage tissue development, whereas the bioreactor vessel provided a permissive microenvironment for chondrogenesis . This work demonstrates the promise of using tissue engineered constructs for in vitro studies of cell interactions and differentiation. Int J Artif Organs, 1998 Jan, 21(1), 43 - 8 Positive biochemical effects of a bioartificial liver support system (BALSS) in a porcine fulminant hepatic failure (FHF) model; Sheil AG et al.; This study describes biochemical changes in the plasma and blood of pigs with devascularised livers treated in a bioartificial liver support system (BALSS) . Porcine hepatic cells were incubated with collagen-coated dextran microspheres (CDM) for 3 hours and the medium tested to determine cellular metabolic activity . Incubation continued for a further 18 hours during which the hepatic cells attach to the CDM . The CDM-attached cells were inoculated into a hollow fibre bioreactor which was part of an extracorporeal support system . Hepatic cell content of the bioreactor was 6 x 10(9) cells . The system was tested in a controlled trial in pigs prepared in a surgical model of fulminant hepatic failure (FHF) . When plasma from FHF pigs was circulated through the device containing hepatic cells, there was significantly less increase in the accumulation of ammonia and most amino acids, together with a decrease in plasma lactate and of one amino acid, compared to control experiments when hepatic cells were excluded . We conclude that primary porcine hepatocytes can contribute beneficial metabolic function in a BALSS. Pharm Dev Technol, 1996 Apr, 1(1), 63 - 8 Novel technology for the preparation of sterile alginate-poly-l-lysine microcapsules in a bioreactor; Abraham SM et al.; The purpose of this study was to develop a method that may be suitable for the commercial manufacture of sterile alginate-polylysine-alginate microcapsules in a bioreactor . A Turbotak atomizing device in conjunction with a Bellco Bioreactor was used to prepare sterile microcapsules . Aseptic procedures were followed using sterilized equipment and materials . Sodium alginate solution was sprayed into calcium chloride solution using the Turbotak, with nitrogen as the atomizing gas . The resultant gelled alginate microcapsules were coated with polylysine and alginate to produce alginate-poly-l-lysine microcapsules . In-process contamination of the atomizing gas and microcapsules was investigated using modified USP sterility tests . Microcapsule size was determined using a light blockage technique (Accusizer) which measures both number and volume weighted mean diameters . The microcapsules prepared passed a modified USP sterility test, and the Bellco Bioreactor was found to minimize the possibilities of environmental contamination and therefore enhanced operator safety . The flow rate of the atomizing gas was determined to significantly alter number and volume weighted mean microcapsule diameters . Statistical analysis indicated that the number weighted mean diameters in conjunction with the volume weighted mean diameters can be used to detect batch-to-batch changes in microcapsule diameters . In conclusion, the modified Bellco Bioreactor offers a novel approach for producing sterile alginate-polylysine microcapsules on a laboratory scale. Br Med Bull, 1997, 53(4), 730 - 44 Bioartificial liver support: developments in hepatocyte culture and bioreactor design; Riordan S et al.; Bioartificial liver devices aim to support patients with acute liver failure (ALF) until orthotopic liver transplantation (OLT) or spontaneous recovery due to hepatic regeneration can occur . However, initial clinical experiences with two devices have indicated that functional efficacy in this setting may be less than in the experimental situation . Several fundamental issues remain unresolved, including the cell mass required to provide meaningful support and which of those hepatocyte components and bioreactor designs so far proposed is best able to do this . In particular, further studies of the efficacy of devices incorporating human hepatocyte lines transformed by either cultural conditions or genetic engineering and those based on multi-channel or flat bed bioreactor designs in which hepatocytes are co-cultured with non-parenchymal cells are awaited . Controlled trials on a multicentre basis in well-defined patient groups and with standardised outcome measures will be required to properly evaluate the clinical value of these devices . A better understanding of factors promoting recovery and regeneration of the native liver and to what extent these can be provided by extracorporeal devices will be essential to the further development of effective bioartificial liver support systems. Hybridoma, 1998 Feb, 17(1), 69 - 72 Generation of murine monoclonal antibodies in serum-free medium; Liu RS et al.; Traditional hybridoma fusion technology requires complete medium with serum supplements to support the growth of hybridoma cells . Serum is also required for subcloning of hybridoma cells to support low density cell growth . IL-6 has been shown to enhance the growth of hybridomas and stimulate antibody production by B cells . We found that the serum requirement in media used for generation of hybridomas can be totally eliminated by substituting with 300 units/ml of IL-6 . Stable hybridoma cell lines were generated to peptide and protein antigens using serum-free adapted P3.653 myelomas as the fusion partner and medium containing IL-6 . Our results indicate that, in general, the fusion efficiencies of serum-free IL-6 supplemented fusions are lower than the fusions employing serum containing media (40%-60% vs . 80%-100%) . However, in spite of the lower fusion efficiency, the number of antigen-specific clones generated using IL-6 was equal to or greater than fusions using serum supplements . The use of IL-6 instead of serum in the generation of monoclonal antibodies (MAbs) has several advantages . We are able to eliminate the costly need for serum in media by using IL-6 that is prepared in house . In addition, we eliminate the need for time-consuming serum-free adaptation of hybridoma cell lines prior to transfer to hollow fiber bioreactors. Sci Total Environ, 1997 Dec 3, 208(1-2), 49 - 57 Estimation of the environmental contamination by phthalic acid esters leaching from household wastes; Bauer MJ et al.; Phthalic acid esters (PAE) have been used as plasticizers in many products . Here we estimate the contaminating potential of PAE codisposed with domestic wastes . In order to determine the maximum threat to the environment, we analysed several fractions of different household wastes . A maximum of 2.6% of di-(2-ethylhexyl) phthalate (DEHP) was measured in 'compound materials' . More than 90% of all the PAE present in the refuse was due to DEHP . Assuming defined compositions of the refuse we calculated that approx . 1 kg of phthalate esters per ton of dry waste may be codisposed . The actual amount of PAE that could be eluted by the leachate was estimated using laboratory bioreactors which were filled with waste of exactly known composition . Only approx . 1 g of PAE per ton of dry waste was leached . However, the elution of hydrophobic DEHP by the leachate is dependent on the composition of the waste which gave rise to different concentrations of dissolved organic carbon . Because of the experimental setup the amounts of leached PAE represent minimum levels of possible environmental contamination . Hence, there will be a constant output of PAE from unlined municipal landfills for decades. J Biotechnol, 1997 Dec 17, 59(1-2), 39 - 52 Plant cell suspension cultures: some engineering considerations; Kieran PM et al.; Higher plants are the source of a vast array of biochemicals which are used as drugs, pesticides, flavourings and fragrances . For some of these compounds, plant cell culture can provide a potential production alternative to traditional cultivation methods or chemical synthesis routes . Many systems have been patented and the last 20 years have seen considerable industrial and academic interest in the development of large scale cultures to produce pharmaceutically active, high value substances . However, the industrial application of plant cell suspension cultures has, to date, been limited . Commercialisation has essentially been impeded by economic feasibility, arising from both biological and engineering considerations . This paper reviews the commercial development of the technology to date and focuses on the impact of specific engineering-related factors, in particular, the shear sensitivity of plant cell suspension cultures . Evidence of sensitivity to hydrodynamic shear in bioreactors has generally been attributed to the physical characteristics of the suspended cells . Recent studies indicate that shear sensitivity may not be as important, in some cases, as initially anticipated. J Clin Lab Anal, 1998, 12(1), 6 - 13 Production of milligram concentrations of free prostate specific antigen (fPSA) from LNCaP cell culture: difference between fPSA from LNCaP cell and seminal plasma; Wu JT et al.; We have established a procedure for the production of milligrams of free PSA (fPSA) from LNCaP cells derived from a human carcinoma of the prostate . By growing LNCaP cells in a serum-free medium in the presence of a synthetic androgen (R1881) and taking advantage of the special design of the Micro-mouse Hollow Fiber Bioreactor, relatively pure fPSA could be obtained . We found that columns containing either Sephacryl S-100 or S-200 could be used to remove the small amount of bovine serum albumin (BSA) and PSA-alpha 1-antichymotrypsin complex (PSA-ACT) from the preparation . More than 90% of the PSA from LNCaP cell cultures are fPSA . Like fPSA from seminal plasma, two fractions of fPSA differing in protease activity can be separated by DEAE-Sepharose chromatography . Based on the band pattern exhibited on the Western blot following sodium dodecyl sulfate-polyacrylamide electrophoresis separation, fPSA from LNCaP contains more inactive PSA isoforms . This was confirmed by chromatofocusing: the isoelectric point (pl) of the major PSA isoforms from the LNCaP cell culture were higher (6.8 and 6.6) than that (6.4 and 6.1) of fPSA from seminal fluid . We conclude that the LNCaP cell culture is a reliable source for obtaining large quantities of pure fPSA both for the preparation of assay calibrators and controls and for studying the difference in fPSA between benign prostate disease and prostate cancer. Indian J Exp Biol, 1997 Aug, 35(8), 886 - 9 Production of L-phenylacetylcarbinol by free and immobilized yeast cells; Tripathi CK et al.; Production of L-phenylacetylcarbinol (L-PAC) through biotransformation of benzaldehyde by free and immobilized cells of the yeast Saccharomyces cerevisiae has been attempted . L-PAC production was found to be maximum (0.4 microliter/ml) when anaerobically grown free cells were used as biocatalyst during aerobic biotransformation for two hours with magnetically stirred bioreactor . Growth under oxygen limited conditions led to accumulation of higher amount of pyruvate decarboxylase enzyme and co-substrate, pyruvate, resulting in higher L-PAC formation . L-PAC yield was low when biotransformations were carried out anaerobically either for aerobically or anaerobically grown free cells . Free cells were found to be more efficient biocatalyst for L-PAC production, as compared with the immobilized cells, with the investigated benzaldehyde concentration (0.3% v/v) and cell density (17.5% w/v) . The study has explored and indicated the possibility of optimizing the yield of L-PAC by growing the yeast cells under oxygen limited condition for suitable aerobic mode of benzaldehyde biotransformation. Analyst, 1997 Dec, 122(12), 1539 - 41 Enzymic flow injection determination of free L-carnitine in infant formulae; Ferreira IM et al.; Infant formula samples were analysed to determine their free L-carnitine content by using flow injection (FI) with an incorporated immobilised carnitine acetyltransferase bioreactor . The methodology was based on the spectrophotometric determination through its reaction with carnitine acetyltransferase coupled with acetyl coenzyme A (acetyl-CoA) and dithiobenzoate . The merging zones technique was used to minimise acetyl CoA consumption . Linearity was observed over the concentration range 10-80 mg l-1 with L-carnitine as standard (r = 0.9998) and the rate of analysis was 50 h-1 infant formula samples . The enzymic FI method afforded a low RSD (2.2%) . Comparisons were made with other methods of known accuracy . The enzymic reactors were stable for 3 months when used daily at the optimum pH. Ann N Y Acad Sci, 1997 Nov 21, 829, 83 - 96 Treatment of trichloroethene-contaminated water with a fluidized-bed bioreactor; Segar RL Jr et al.; Biological systems are a potentially attractive means of treatment for groundwater contaminated with chlorinated solvents . In this research, a bench-scale fluidized-bed biological reactor (FBBR) was used to treat water contaminated by trichloroethene (TCE) . The bed volume was 2.4 L and the flow rate was 0.8 L/min . Applications of the reactor are primarily for treatment of groundwater extracted during aquifer remediation . Reactor feed water was amended with phenol and nutrients to stimulate the growth of bioparticles with the ability to cometabolize TCE . The FBBR was operated in a bed growth mode for two weeks followed by a TCE removal period of two weeks during which TCE and phenol were fed simultaneously . Experiments were conducted to determine the removal of 0.1 mg/L TCE for phenol loading rates ranging from 3 to 9 mg/min . Higher phenol loading inhibited TCE degradation and resulted in phenol not being completely removed by the reactor . Phenol was not detected in the reactor effluent at loadings below 6 mg/min (3.6 g/L/d) . A phenol to TCE mass ratio of 75:1 provided an average 60% removal of 0.1 mg/L TCE at an empty bed contact time of 3 minutes . Higher removal of TCE may be possible in field-scale reactors where the FBBR height (bed-depth) may be extended. Biologicals, 1997 Dec, 25(4), 415 - 9 A rapid and simple procedure to detect the presence of MVM in conditioned cell fluids or culture media; Chang A et al.; During the manufacture of biopharmaceuticals, numerous adventitious agents have been detected in Master Cell Banks, end-of-production cells as well as bulk harvest fluid . Recently, a number of large-scale production bioreactors have become infected with Minute Virus of Mice (MVM) during cGMP (current good manufacturing practices) operations, and this has resulted in both the loss of product and the need for major cleaning validation procedures to be put in place . We have developed a simple DNA extraction/PCR assay to detect the presence of MVM in cell culture supernatant (conditioned cell fluids) . This highly specific assay can detect 10 or fewer genome equivalents (copies) of MVM following PCR and gel electrophoresis visualization . For routine high-throughput detection, 300-100 copies could be consistently detected . The extraction procedure was shown to reliably detect MVM at a concentration of 1 TCID50/ml . The combination of the extraction/PCR procedure establishes a powerful, sensitive, specific assay that can detect the presence of MVM sequences with a 1-day turnaround time. Int J Artif Organs, 1997 Nov, 20(11), 644 - 9 Immunoisolation of hybrid liver support systems by semipermeable membranes; Gleissner M et al.; Immunoisolation of hybrid liver support systems (LSS) utilizing suitable semipermeable membranes as an immune barrier enables neither immunocompetent cytotoxic factors to cause damage to the hepatocytes in the bioreactor nor xenogenic hepatocyte products to cause immunological side effects in patients . To determine the capability of membranes as an immune barrier, 6 flat membranes were investigated: Cuprophan (C-100), cut-off MW 1000, Cuprophan (C-240), cut-off MW 10,000, Polypropylen hydrophilic and hydrophobic (PPhi, PPho), cut-off MW 500,000-1,000,000, Polysulfon (PS), cut-off MW 1,000,000, Polyamid (PA), cut-off beyond MW 1,000,000 . The permeability of the membranes to plasma factors and liver protein fractions (LP) was studied by routine biochemical methods and gel electrophoresis . In a second study, pigs (n=7) were immunised by LP after membrane passage . The results showed PA, PS, and PPhi to be completely permeable for plasma factors and LP C-100 and C-240 for urophanic substances, and C-240 again for LP under MW 14.000 . All 7 pig sera studied by Western blot discovered pre-formed xenoreactive natural IgG-antibodies (NAB) against human liver antigen (AG) with MW 26.000 . AB de-novo-synthesis was demonstrated for AG with MW 45.000 . No AB-synthesis was induced for epitopes under MW 26,000 . These results suggest that limiting the cut-off of bioreactor outflow membranes to MW < 26,000 could avoid immunological side effects to patients. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13885 - 90 Tissue engineering of cartilage in space; Freed LE et al.; Tissue engineering of cartilage, i.e., the in vitro cultivation of cartilage cells on synthetic polymer scaffolds, was studied on the Mir Space Station and on Earth . Specifically, three-dimensional cell-polymer constructs consisting of bovine articular chondrocytes and polyglycolic acid scaffolds were grown in rotating bioreactors, first for 3 months on Earth and then for an additional 4 months on either Mir (10(-4)-10(-6) g) or Earth (1 g) . This mission provided a unique opportunity to study the feasibility of long-term cell culture flight experiments and to assess the effects of spaceflight on the growth and function of a model musculoskeletal tissue . Both environments yielded cartilaginous constructs, each weighing between 0.3 and 0.4 g and consisting of viable, differentiated cells that synthesized proteoglycan and type II collagen . Compared with the Earth group, Mir-grown constructs were more spherical, smaller, and mechanically inferior . The same bioreactor system can be used for a variety of controlled microgravity studies of cartilage and other tissues . These results may have implications for human spaceflight, e.g., a Mars mission, and clinical medicine, e.g., improved understanding of the effects of pseudo-weightlessness in prolonged immobilization, hydrotherapy, and intrauterine development. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13554 - 8 A direct electrode-driven P450 cycle for biocatalysis; Reipa V et al.; The large potential of redox enzymes to carry out formation of high value organic compounds motivates the search for innovative strategies to regenerate the cofactors needed by their biocatalytic cycles . Here, we describe a bioreactor where the reducing power to the cycle is supplied directly to purified cytochrome CYP101 (P450cam; EC 1.14.15.1) through its natural redox partner (putidaredoxin) using an antimony-doped tin oxide working electrode . Required oxygen was produced at a Pt counter electrode by water electrolysis . A continuous catalytic cycle was sustained for more than 5 h and 2,600 enzyme turnovers . The maximum product formation rate was 36 nmol of 5-exo-hydroxycamphor/nmol of CYP101 per min. Biotechnol Prog, 1997 Sep-Oct, 13(5), 583 - 9 Biodesulfurization of flue gases and other sulfate/sulfite waste streams using immobilized mixed sulfate-reducing bacteria; Selvaraj PT et al.; Sulfur dioxide (SO2) is one of the major pollutants in the atmosphere that cause acid rain . Microbial processes for reducing SO2 to hydrogen sulfide (H2S) have previously been demonstrated by utilizing mixed cultures of sulfate-reducing bacteria (SRB) with municipal sewage digest as the carbon and energy source . To maximize the productivity of the bioreactor for SO2 reduction in this study, various immobilized cell bioreactors were investigated: a stirred tank with SRB flocs and columnar reactors with cells immobilized in either potassium-carrageenan gel matrix or polymeric porous BIO-SEP beads . The maximum volumetric productivity for SO2 reduction in the continuous stirred-tank reactor (CSTR) with SRB flocs was 2.1 mmol of SO2/(h.L) . The potassium-carrageenan gell matrix used for cell immobilization was not durable at feed sulfite concentrations greater than 2000 mg/L (1.7 mmol/(h.L)) . A columnar reactor with mixed SRB cells that had been allowed to grow into highly stable BIO-SEP polymeric beads exhibited the highest sulfite conversion rates, in the range 16.5 mmol/(h.L) (with 100% conversion) to 20 mmol/(h.L) (with 95% conversion) . The average specific activity for sulfite reduction in the column, in terms of dry weight of SRB biomass, was 9.5 mmol of sulfite/(h.g) . In addition to flue gas desulfurization, potential applications of this microbial process include the treatment of sulfate/sulfite-laden wastewater from the pulp and paper, petroleum, mining, and chemical industries. Appl Microbiol Biotechnol, 1997 Dec, 48(6), 745 - 52 Bioremediation of pentachlorophenol-contaminated soil by bioaugmentation using activated soil; Barbeau C et al.; The use of an indigenous microbial consortium, pollutant-acclimated and attached to soil particles (activated soil), was studied as a bioaugmentation method for the aerobic biodegradation of pentachlorophenol (PCP) in a contaminated soil . A 125-l completely mixed soil slurry (10% soil) bioreactor was used to produce the activated soil biomass . Results showed that the bioreactor was very effective in producing a PCP-acclimated biomass . Within 30 days, PCP-degrading bacteria increased from 10(5) cfu/g to 10(8) cfu/g soil . Mineralization of the PCP added to the reactor was demonstrated by chloride accumulation in solution . The soil-attached consortium produced in the reactor was inhibited by PCP concentrations exceeding 250 mg/l . This high level of tolerance was attributed to the beneficial effect of the soil particles . Once produced, the activated soil biomass remained active for 5 weeks at 20 degrees C and for up to 3 months when kept at 4 degrees C . The activated attached soil biomass produced in the completely mixed soil slurry bioreactor, as well as a PCP-acclimated flocculent biomass obtained from an air-lift immobilized-soil bioreactor, were used to stimulate the bioremediation of a PCP-impacted sandy soil, which had no indigenous PCP-degrading microorganisms . Bioaugmentation of this soil by the acclimated biomass resulted in a 99% reduction (from 400 mg/kg to 5 mg/kg in 130 days) in PCP concentration . The PCP degradation rates obtained with the activated soil biomass, produced either as a biomass attached to soil particles or as a flocculent biomass, were similar. Artif Organs, 1997 Nov, 21(11), 1177 - 81 Semipermeable hollow fiber membranes in hepatocyte bioreactors: a prerequisite for a successful bioartificial liver? Flendrig LM, te Velde AA, Chamuleau RA. Recent studies have shown that liver support systems based on viable hepatocytes can prolong life in animal models of acute liver failure . Now the time has come to elucidate the design characteristics that are essential to construct an efficient bioreactor . The gold standard remains the intact liver . Despite the very high cell density in this organ, individual cell perfusion is guaranteed resulting in low diffusional gradients which are essential for optimal mass transfer . These conditions are not met in bioreactors based on hollow fiber membranes . Moreover, the semipermeable membranes can foul and act as a diffusional barrier between the hepatocytes and the blood or plasma of the recipient . We devised a novel bioreactor for use as a bioartificial liver that does not include hollow fiber membranes for blood or plasma perfusion . The device is based on an integral oxygenator and a nonwoven polyester matrix material for hepatocyte culture as small aggregates . The efficacy of this original design was tested in rats with liver ischemia . Preliminary results show statistically significantly improved survival; life was prolonged 100% compared to the control experiments. Folia Microbiol (Praha), 1997, 42(4), 349 - 52 Characterization of phytase produced by Aspergillus niger; Dvorakova J et al.; The extracellular activity of Aspergillus niger phytase at the end of the growth phase was 132 nkat/mL in a laboratory bioreactor . The purified enzyme has molar mass approximately 100 kDa, pH optimum at 5.0, temperature optimum at 55 degrees C and high pH and temperature stability . The Km for dodecasodium phytate, calcium phytate and 4-nitrophenyl phosphate are 0.44, 0.45 and 1.38 mmol/L, respectively . The enzyme is noncompetively inhibited by inorganic monophosphate (Ki = 2.85 mmol/L) and by Cu2+, Zn2+, Hg2+, Sn2+, Cd2+ ions and strongly by F- ones; it is activated by Ca2+, Mg2+ and Mn2+ ions . The substrate specificity of phytase is broad with the highest affinity to calcium phytate. J Immunol Methods, 1997 Oct 13, 208(1), 65 - 73 Single-step purification of bispecific monoclonal antibodies for immunotherapeutic use by hydrophobic interaction chromatography; Manzke O et al.; A method for large scale production and single-step purification of bispecific antibodies is described . Hybrid-hybridomas were grown in hollow-fibre bioreactors with an average yield of 8 to 12 g of immunoglobulin per month . Bispecific antibodies were purified from the bioreactor supernatant by hydrophobic interaction chromatography which resolves bispecific antibodies, monospecific immunoglobulins, and culture medium supplements in one single chromatographic step . Proteins were analyzed by ELISA, SDS-PAGE, isoelectric focussing, indirect fluorescence staining, CTL-stimulation and T-cell proliferation assays . Finally, antibody preparations were checked for the presence of endotoxin and mouse DNA . Our results suggest that functional bispecific antibodies for use in therapeutic applications can be batch purified from bioreactor harvest by hydrophobic interaction chromatography in a single step . Compared to other methods such as affinity chromatography (protein A/G), ion-exchange or hydroxyapatite chromatography, our protocol offers a substantial reduction in labor time, cost, protein loss, and risk of contamination. Chin J Biotechnol, 1997, 13(3), 169 - 76 Extractive L-lactic acid fermentation with immobilized Rhizopus oryzae in a three-phase fluidized bed; Lu Y et al.; The Rhizopus oryzae was immobilized by the calcium alginate entrapment method . A three-phase fluidized-bed bioreactor was designed to perform the immobilized-cell L-lactic acid fermentation . A solvent extraction column was coupled with the bioreactor to remove L-lactic acid from the fermentation broth . The TRPO was selected as solvent and sulfonated kerosene as diluent . The results indicated that the pH value in the broth was regulated above pH 3.5 and the fermentation rate was as high as 11 g L-lactic acid per hour per liter of beads . A mathematical model was proposed to describe the concentration of L-lactic acid in the extractive fermentation. Biotechnol Appl Biochem, 1997 Dec, 26 ( Pt 3), 203 - 12 Immobilization of L-asparaginase into a biocompatible poly(ethylene glycol)-albumin hydrogel: evaluation of performance in vivo; Jean-Francois J et al.; The L-asparaginase of Escherichia coli (ASNase) is currently used in combination with antineoplastic drugs to treat various lymphoblastic leukaemias . However, its use is limited by severe immunological reactions and the short serum half-life associated with the enzyme . Immobilization of ASNase into a biocompatible matrix can greatly decrease the immunogenicity of the enzyme, increase its half-life in vivo and its therapeutic index . Thus the E . coli ASNase was immobilized in a biocompatible hydrogel made of rat serum albumin and poly(ethylene glycol) (PEG; molecular mass 10 kDa) . The effectiveness of this enzymic bioreactor to deplete serum L-asparagine was evaluated after its peritoneal implantation in rats . Seven units of immobilized ASNase/rat depleted serum asparagine to an undetectable level (< 1 microM) during 6 days, while 5 units of immobilized ASNase/rat decreased the level of serum asparagine by 85-90% during at least 2 days . Under both conditions asparagine levels returned to normal about 10 days after surgery, and hydrogels still retained 80% of their enzymic activity when assayed in vitro . After 10-14 days in vivo, hydrogels became opaque and surrounded by a fibrotic capsule with a few inflammatory sites . Nevertheless, the enzymic hydrogel showed great stability in vivo, and, after 4 months of implantation, 12% of the initial ASNase activity was still present . At 6 months, histological analysis showed stabilization of the fibrotic capsule thickness . Assays on the levels of ASNase and asparagine synthetase indicated an induction of the latter activity, mainly in the pancreas when compared with the level observed in spleen or liver . ELISA tests at 28 days and 120 days showed the presence of anti-ASNase (and, in lower amounts, anti-PEG) antibodies in sera of implanted rats . As observed with other enzyme-immobilization systems used in vivo, the formation of fibroblast-like cell layers around the implant, which block the translocation of the substrate into the enzymic matrix, is the major factor affecting the performance and longevity of the bioreactor. Anal Chem, 1997 Nov 15, 69(22), 4601 - 7 Improving the activity of immobilized subtilisin by site-specific attachment to surfaces; Huang W et al.; Understanding the properties of immobilized proteins is critical to the optimal design of biosensors, bioseparations, and bioreactors . The protease subtilisin BPN' was used as a model protein to study how the orientation of immobilized enzyme molecules on surfaces affects their catalytic properties . To achieve this goal, a single cysteine residue was introduced into the cysteine-free enzyme by site-directed mutagenesis . This cysteine residue was designed to be away from the active site of the enzyme . The enzyme molecules were immobilized through the side-chain sulfhydryl group of the cysteine residue on several supports . This site-specific immobilization method leads to ordered two-dimensional arrays of enzyme molecules on the support surface with the active sites of the enzyme oriented toward the solution phase . Such oriented immobilized subtilisin demonstrated a higher catalytic efficiency compared to subtilisin that was immobilized by a conventional method that leads to random immobilization. Biotechnol Prog, 1997 Nov-Dec, 13(6), 810 - 3 Enzymatic large-scale production of 2-keto-3-deoxy-D-glycero-D-galacto-nonopyranulosonic acid in enzyme membrane reactors; Salagnad C et al.; The enzymatic synthesis of 2-keto-3-deoxy-D-glycero-D-galacto-nonopyranulosonic acid (KDN) starting from D-mannose and pyruvic acid using Neu5Ac-aldolase has been scaled up . A repetitive batch ultrafiltration bioreactor was used for the KDN synthesis on 100 g scale with a conversion of up to 85% . Furthermore, a 440 mL pilot-scale enzyme membrane reactor (EMR) was performed for the continuous production of KDN . Conversion of mannose was 75% at a space--time yield of 375 g/(L d) . KDN was advanteageously isolated by crystallization with an overall yield of 75%. Biotechnol Prog, 1997 Nov-Dec, 13(6), 799 - 804 Kinetics of growth and ribosome-inactivating protein production from Trichosanthes kirilowii plant cell cultures in a 5-L bioreactor; Stoner MR et al.; Ribosome-inactivating proteins, named for their ability to inhibit protein translation in cell-free systems, are an important class of natural plant defense proteins with potential human therapeutic and agricultural applications . The kinetics of growth, nutrient consumption, and extracellular protein translation inhibitory activity are presented for Trichosanthes kirilowii plant cell suspensions in 5-L bioreactors at two agitation rates (50 and 100 rpm) . The cultures had a 7-9.5 day lag phase followed by exponential growth with a doubling time of less than 2 days . Biomass concentrations reached levels of approximately 19 g (dry weight)/L . Protein translation inhibitory activity was observed in the culture broths during the exponential growth phase and reached levels of approximately 50-60 units . No detrimental effects of agitation were observed at 100 rpm . These studies demonstrate the potential for plant cell culture production of ribosome-inactivating proteins in bioreactor systems. Am J Kidney Dis, 1997 Nov, 30(5 Suppl 4), S28 - 31 The bioartificial renal tubule assist device to enhance CRRT in acute renal failure; Humes HD et al.; Current therapy for acute tubular necrosis (ATN) continues to have an exceedingly high mortality rate, exceeding 50% even with dialytic or hemofiltrative support . Current renal replacement therapy in ATN only substitutes for filtration function of the kidney but not its cellular metabolic functions . Replacing these metabolic functions may optimize current therapy for this devastating disease process . In this regard, a renal tubule assist device (RAD) has been developed to be placed in an extracorporeal continuous hemoperfusion circuit in series with a hemofilter . The RAD consists of porcine renal proximal tubule cells grown as confluent monolayers of a multifiber bioreactor with a membrane surface area from 0.4 to 1.6 m2 . The cells along the inner surface of the hollow fibers are immunoprotected from the patient's blood by the hollow fiber membrane . In preliminary experiments in uremic dogs, this device has been shown to tolerate a uremic environment while providing reabsorptive, metabolic, and endocrinologic activity . Pilot human trials of the RAD are anticipated within the next year to improve current renal replacement therapy in ATN. Braz J Med Biol Res, 1997 Aug, 30(8), 923 - 8 Large-scale production and purification of recombinant protein from an insect cell/baculovirus system in Erlenmeyer flasks: application to the chicken poly(ADP-ribose) polymerase catalytic domain; Miranda EA et al.; A simple and inexpensive shaker/Erlenmeyer flask system for large-scale cultivation of insect cells is described and compared to a commercial spinner system . On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive bioreactor consisting of Erlenmeyer flasks and an ordinary shaker waterbath . Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 x 10(9) infected cells in three liters of culture. Curr Microbiol, 1997 Dec, 35(6), 356 - 62 Stable Transformation of Chlorella: Rescue of Nitrate Reductase-Deficient Mutants with the Nitrate Reductase Gene Dawson HN, Burlingame R, Cannons AC. Unicellular green algae, like Chlorella, offer a potentially useful system for the expression of heterologous proteins . However, the development of Chlorella as a bioreactor has been delayed owing to the lack of a stable transformation technique . Here we report on the use of micro-projectile bombardment to introduce the nitrate reductase (NR) gene from Chlorella vulgaris into NR-deficient Chlorella sorokiniana mutants, resulting in stable transformants . The stable transformants were able to grow on nitrate medium after repeated passages between selective and nonselective medium and exhibited inducible nitrate reductase activity comparable to that of wild-type cells . Southern analysis suggests homologous recombination occurs with insertion of the wild type gene into the mutated gene and that the genes of the two Chlorellaspecies used are very similar . Specific RNase protection assays, selecting for a poorly conserved region of the gene, identified the presence of the C . vulgaris NR transcript only in the transformed C . sorkiniana mutant and not in the mutant. Appl Microbiol Biotechnol, 1997 Sep, 48(3), 424 - 30 Removal of tetrachloroethylene in an anaerobic column bioreactor; Noftsker C et al.; Removal of tetrachloroethylene (perchloroethylene; C2Cl4) by microbial consortia from two sites with different C2Cl4 exposure histories was examined in a bench-scale anaerobic column bioreactor . It was hypothesized that optimal removal would be observed in the reactor packed with sediments having an extensive exposure history . Microbial consortia were enriched from hyporheic-zone (HZ) sediments from the Portneuf aquifer near Pocatello, Idaho, and from industrial-zone (IZ) sediments from a highly contaminated aquifer in Portland, Oregon . Lactate and acetate were the electron donors during experiments conducted over 9 and 7 months for HZ and IZ sediments, respectively . In the HZ bioreactor, the retention time ranged from 31 h to 81 h, and inlet C2Cl4 concentrations ranged from 0.1 ppm to 1.0 ppm . Dechlorination of C2Cl4 averaged 60% and reached a maximum of 78% . An increase in C:N from 27:1 to 500:1 corresponded to an 18% increase in removal efficiency . Trichloroethylene production corresponded to decreased effluent C2Cl4; further intermediates were not detected . In the IZ bioreactor, the retention time varied from 34 h to 115 h; the inlet C2Cl4 concentration was 1.0 ppm . C2Cl4 removal averaged 70% with a maximum of 98% . Trichloroethylene and cis-dichloroethylene were detected in the effluent . Increases in C:N from 50:1 to 250:1 enhanced dechlorination activity. Appl Environ Microbiol, 1997 Oct, 63(10), 4090 - 2 Effect of simulated microgravity and shear stress on microcin B17 production by Escherichia coli and on its excretion into the medium; Fang A et al.; Production of the antibacterial polypeptide microcin B17 (MccB17) by Escherichia coli ZK650 was inhibited by simulated microgravity . The site of MccB17 accumulation was found to be different, depending on whether the organism was grown in shaking flasks or in rotating bioreactors designed to establish a simulated microgravity environment . In flasks, the accumulation was cellular, but in the reactors, virtually all the microcin was found in the medium . The change from a cellular site to an extracellular one was apparently not a function of gravity, since extracellular production occurred in these bioreactors, irrespective of whether they were operated in the simulated microgravity or normal gravity mode . More probably, excretion is due to the much lower degree of shear stress in the bioreactors . Addition of even a single glass bead to the 50-ml medium volume in the bioreactor created enough shear to change the site of MccB17 accumulation from the medium to the cells. Cell Transplant, 1997 Sep-Oct, 6(5), 537 - 40 An attempt to add biological functions by genetic engineering in order to produce high-performance bioreactor cells for hybrid artificial liver: transfection of glutamine synthetase into Chinese hamster ovary (CHO) cell; Enosawa S et al.; In the course of immortalization, hepatocyte cell lines lose their original differentiated functions, such as ammonia removal and urea formation, drug metabolism, serum protein synthesis, etc . (Enosawa et al., Cell Transplant . 5:S39-S40; 1996) . With the aim of adding lost or deficient functions and producing cell lines for the bioreactor of a hybrid artificial liver, rat glutamine synthetase (GS) gene was transfected into Chinese hamster ovary (CHO) cells, because it is able to lower the ammonia level . The GS gene-inserted pSV2 plasmid was transfected into the CHO-K1 line by electroporation . Transfected CHO (GS-CHO) cells were cultured in a glutamine-free medium containing ammonia, glutamic acid, and the GS inhibitor methionine sulfoximine (MSX) . The MSX concentration was increased stepwise from 25 mumol/L to 1600 mumol/L to amplify the GS gene . In several GS-CHO sublines resistant to 300-1600 mumol/L of MSX, the specific activities of GS were increased from 0.2 x 10(4) to 1.7-2.9 x 10(4) unit/10(6) cells . When the amplified GS-CHO cells were cultured in the ammonia-containing medium, a slow but steady decrease of the ammonia level was observed when the level was high . Finally, the prospect of genetically modulated cells for bioreactors in the hybrid artificial liver is discussed. Mol Reprod Dev, 1997 Nov, 48(3), 324 - 31 Cre-mediated recombination at the murine whey acidic protein (mWAP) locus; Rucker EB et al.; The mouse whey acidic protein (WAP) gene in mouse embryonic stem (ES) cells has been targeted with a loxP-flanked neomycin phosphotransferase-thymidine kinase (neo-TK) cassette inserted into exon 4 . Southern blot revealed that 51 of 199 colonies were correctly targeted (1:4) . Next, a Cre-encoding plasmid was electroporated into a targeted cell line to cause the deletion of the neo-TK cassette . Modified ES cell colonies were identified by polymerase chain reaction (PCR); 44 out of 50 colonies (88%) had undergone Cre-mediated deletion . Finally, a loxP-tagged cell line was co-electroporated with a Cre-encoding plasmid and a loxP-containing neo plasmid for site-specific insertion into the WAP locus . The frequency of this event was 23% (11 of 48) of that obtained with random integration . This demonstrates the feasibility of using the Cre-loxP system for site-specific integration in ES cells . Moreover, this is the first report of targeting a loxP-containing transgene into a predetermined location in ES cells . Ultimately, a mouse model derived from these modified ES cells will usher in a second generation of animal "bioreactor" models where the inserted transgene is controlled exclusively by the endogenous locus regulatory elements . In addition, oncogenesis can be explored from single copy oncogene/tumor suppressor gene inserts, which are regulated in a temporal and tissue-specific manner . It is hoped that regulation of transgene expression in this fashion will help elucidate the underlying mechanisms of normal development in the mammary gland. Enzyme Microb Technol, 1997 Oct, 21(5), 355 - 60 The influence of pH and aeration rate on the fermentation of D-xylose by Candida shehatae; Sanchez S et al.; The effects of the initial pH and air supply on the production of ethanol from D-xylose using the yeast Candida shehatae in a batch reactor were investigated . The initial pH was altered within the range of 2.5-6.5 and the specific aeration rate from 0.0-0.3 vv-1 min-1 . The results showed that the most favorable initial pH for ethanol production was 4.5 and aeration via the stirring vortex of the bioreactor was sufficient . Under these conditions, the maximum specific growth rate (mu(m)) was 0.329 h-1; biomass production rate (b), 0.024 kg m-3 h-1; overall biomass yield (YGx/s), 0.036 kg kg-1; the specific uptake rate of D-xylose (qs), 2.0 kg kg-1 h-1; and the specific ethanol production rate (qE), 0.72 kg kg-1 h-1 (both at 20 h culture time) . The average xylitol yield (Yxy/s) was 0.078 kg kg-1 and the overall ethanol yield (YGE/s), 0.41 kg kg-1 . Both qs and qE diminished once the exponential growth phase was over. J Dairy Sci, 1997 Sep, 80(9), 2213 - 24 Transgenic dairy cattle: genetic engineering on a large scale; Wall RJ et al.; Amid the explosion of fundamental knowledge generated from transgenic animal models, a small group of scientists has been producing transgenic livestock with goals of improving animal production efficiency and generating new products . The ability to modify mammary-specific genes provides an opportunity to pursue several distinctly different avenues of research . The objective of the emerging gene "pharming" industry is to produce pharmaceuticals for treating human diseases . It is argued that mammary glands are an ideal site for producing complex bioactive proteins that can be cost effectively harvested and purified . Consequently, during the past decade, approximately a dozen companies have been created to capture the US market for pharmaceuticals produced from transgenic bioreactors estimated at $3 billion annually . Several products produced in this way are now in human clinical trials . Another research direction, which has been widely discussed but has received less attention in the laboratory, is genetic engineering of the bovine mammary gland to alter the composition of milk destined for human consumption . Proposals include increasing or altering endogenous proteins, decreasing fat, and altering milk composition to resemble that of human milk . Initial studies using transgenic mice to investigate the feasibility of enhancing manufacturing properties of milk have been encouraging . The potential profitability of gene "pharming" seems clear, as do the benefits of transgenic cows producing milk that has been optimized for food products . To take full advantage of enhanced milk, it may be desirable to restructure the method by which dairy producers are compensated . However, the cost of producing functional transgenic cattle will remain a severe limitation to realizing the potential of transgenic cattle until inefficiencies of transgenic technology are overcome . These inefficiencies include low rates of gene integration, poor embryo survival, and unpredictable transgene behavior. Appl Microbiol Biotechnol, 1997 Aug, 48(2), 198 - 203 The effect of oxidative stress on the production of the recombinant protein, interferon gamma, produced by Chinese hamster ovary cells in stirred-batch culture; Dunster CA et al.; The CHO320 cell line, engineered to produce human interferon gamma was investigated with regard to its susceptibility to oxidative stress . Batch cultures of the cells grown in a bench-top bioreactor exhibited no marked response to changes in oxygen concentration between 6% and 14% whereas cell growth and recombinant protein production were inhibited by increasing the oxygen to 20% . High concentrations of hydrogen peroxide (in excess of 200 microM) were required to inhibit growth of the CHO320 cells whereas concentrations of 50 microns and 100 microM had no effect on recombinant protein production . Buthionine sulphoximine (50 microM and 100 microM) completely depleted the cells of glutathione within 24 h; however, no quantitative effect on recombinant protein production was seen . It is concluded that the CHO320 cells are, possibly as a consequence of the long selection process they have undergone, very resistant to oxidative stress. Cell Biol Toxicol, 1997 Jul, 13(4-5), 357 - 64 Characterization of the three-compartment gel-entrapment porcine hepatocyte bioartificial liver; Sielaff TD et al.; A hybrid bioartificial liver device supporting a large mass of cells expressing differentiated hepatocyte metabolic capabilities is necessary for the successful treatment of fulminant hepatic failure . The three-compartment gel-entrapment porcine hepatocyte bioartificial liver was designed to provide "bridge" support to transplantation or until native liver recovery is achieved for patients with acute liver failure . The device is an automated mammalian cell culture system supporting 6-7 x 10(9) porcine hepatocytes entrapped in a collagen matrix and inoculated into the capillary lumen spaces of two 100 kDa molecular mass cut-off hollow fiber bioreactors . Gel contraction recreates a small lumen space within the hollow fiber which allows for the delivery of a nutrient medium . This configuration supported hepatocyte viability and differentiated phenotype as measured by albumin synthesis, ureagenesis, oxygen consumption, and vital dye staining during both cell culture and ex vivo application . The hollow fiber membrane was also shown to isolate the cells from xenogenic immunoglobulin attack . The gel-entrapment bioartificial liver maintained a large mass of functional hepatocytes by providing a three-dimensional cell culture matrix, by delivering basal nutrients through lumen media perfusion, and by preventing rejection of the xenocytes . These features make this device a favorable candidate for the treatment of clinical fulminant hepatic failure. Cell Biol Toxicol, 1997 Jul, 13(4-5), 349 - 55 Long-term liver cell cultures in bioreactors and possible application for liver support; Gerlach JC; Hybrid artificial liver systems are being developed as a temporary extracorporeal liver support therapy . A short overview is given which emphasizes the development of hepatocyte culture models for bioreactors, subsequent in vitro studies, animal studies and the clinical application of hybrid liver support systems . An own bioreactor construction has been designed for the utilization of hepatocytes and sinusoidal endothelial cells . The reactor is based on capillaries for hepatocyte aggregate immobilization, coated with biomatrix . Four separate capillary membrane systems, each permitting a different function, are woven in order to create a three-dimensional network . Cells are perfused via independent capillary membrane compartments . Decentralized oxygen supply and carbon dioxide removal with low gradients is possible . There is a decentralized co-culture compartment for nonparenchymal liver cells . The use of identical parallel units to supply a few hepatocytes facilitates scale-up. J Immunol Methods, 1997 Jul 14, 205(2), 109 - 14 Measurement of glucose consumption by hybridoma cells growing in hollow fiber cartridge bioreactors: use of blood glucose self-monitoring devices; Nayak RC et al.; Two different types of blood glucose self-monitoring device, designed for use by diabetics (Accu-Chek Advantage* and Accu-Chek III*), were evaluated for the purpose of monitoring glucose concentration in tissue culture media . The Accu-Chek Advantage* meter was found to systematically overestimate the glucose concentration in a variety of commonly used tissue culture media by 50-90%, in comparison with their formulated glucose concentration . The Accu-Chek III* meter reliably estimated glucose concentrations from 100 to 300 mg/dl and overestimated glucose concentrations above 300 mg/dl . The systematic overestimation of glucose concentration in tissue culture fluids by the Accu-Chek Advantage* meter was further investigated . A standard curve was constructed and the meter reading was found to be linearly related to the actual glucose concentration and the best linear fit was given by the formula y = -43.504 + 1.9246x, where y is the meter reading and x is the actual glucose concentration . Rearranging the equation to make x the subject gave the following algorithm x = (y + 43.504) divided by 1.9246 which could be used to correct the 'raw' meter reading . The mean corrected glucose concentrations deviated from the formulated glucose concentration by less than 3.5% in five media tested, indicating that this meter is more than adequate for monitoring glucose consumption by cells growing in hollow fiber cartridge bioreactors, when used in conjunction with this correction factor. Appl Microbiol Biotechnol, 1997 Jul, 48(1), 47 - 52 Mannitol dehydrogenase from Rhodobacter sphaeroides Si4: subcloning, overexpression in Escherichia coli and characterization of the recombinant enzyme; Schafer A et al.; By polymerase chain reaction mutagenesis techniques, an NdeI restriction site was introduced at the initiation codon of the mannitol dehydrogenase (MDH) gene (mtlK) of Rhodobacter sphaeroides Si4 . The mtlK gene was then subcloned from plasmid pAK74 into the NdeI site of the overexpression vector pET24a+ to give plasmid pASFG1 . Plasmid pASFG1 was introduced into Escherichia coli BL21(DE3), which was grown in a 1.5-1 bioreactor at 37 degrees C and pH 7.0 . Overexpression of MDH in Escherichia coli BL21(DE3) {pASFG1} was determined by enzymatic analysis and sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis . Under standard growth conditions, E . coli produced considerable amounts of a polypeptide that correlated with MDH in SDS gels, but the activity yield was low . Decreasing the growth temperature to 27 degrees C and omitting pH regulation resulted in a significant increase in the formation of soluble and enzymatically active MDH up to a specific activity of 12.4 U/mg protein and a yield of 26,000 U/l, which corresponds to 0.38 g/l MDH . This was an 87-fold overexpression of MDH compared to that of the natural host R . sphaeroides Si4, and a 236-fold improvement of the volumetric yield . MDH was purified from E . coli BL21(DE3) {pASFG1} with 67% recovery, using ammonium sulfate precipitation, hydrophobic interaction chromatography, and gel filtration . Partial characterization of the recombinant MDH revealed no significant differences to the wild-type enzyme. Transfusion, 1997 Jul, 37(7), 685 - 90 Retroviral transduction of peripheral blood leukocytes in a hollow-fiber bioreactor; Shankar R et al.; BACKGROUND: Peripheral blood white cells (leukocytes) (PBLs) have been used as effective targets for genetic manipulation by transduction with retroviruses in open systems . A semi-closed hollow-fiber bioreactor was tested for culturing and transducing lymphocytes . STUDY DESIGN AND METHODS: PBLs were isolated from five healthy donors, and 5 x 10(7) cells were cultured in hollow-fiber bioreactors for 4 days after stimulation with anti-CD3 in medium containing 200 units per mL of recombinant interleukin 2 . Transduction with retroviral vector containing the gene for iduronate-2-sulfatase and G 418 resistance, L2SN, was performed daily on Days 4, 5, 6, and 7, and the cells were expanded for an additional 3 days . RESULTS: PBLs from three donors were harvested from the bioreactor after transduction and expansion, and 4.5 x 10(9), 7.0 x 10(9), and 2.9 x 10(9) cells were recovered, representing 90-, 136-, and 58-fold expansions . The transduction frequency of L2SN was 10, 5, and 1 percent, respectively . For additional expansion of PBLs, in two cases the bioreactor was reinoculated with 5 x 10(7) cells, which were expanded again for 16 and 8 days, respectively, yielding 1.4 x 10(9) and 3.1 x 10(9) cells, which reflected an additional 28- and 62-fold expansion of cells . PBLs from two other donors were transduced and expanded in the bioreactor, and then 0.8 mg per mL of G 418 was added to the medium in an attempt to enrich the transduced population . Although 2.5 and 10 percent of the cells were transduced, cell death and absence of expansion in the presence of G 418 resulted in final cell lots with viabilities of only 4 and 8 percent . In all cases, the harvested cells tested negative in bacterial and fungal cultures . CONCLUSION: Hollow-fiber bioreactors are an efficient and effective system for the retroviral transduction and expansion of PBLs for clinical gene therapy. Thromb Haemost, 1997 Jul, 78(1), 543 - 7 The past, present and future of transgenic bioreactors; Drohan WN; Hybrid genes can control the tissue-specific synthesis of human proteins in transgenic animals . Thus, it is now possible to produce proteins of biomedical value in the body fluids or cells of transgenic livestock . In fact, the first transgenically produced protein, antithrombin III, is now in clinical trials and others will soon follow. Gene Ther, 1997 Jun, 4(6), 600 - 10 Novel retroviral packaging cell lines: complementary tropisms and improved vector production for efficient gene transfer; Forestell SP et al.; We report increased transduction of human hematopoietic progenitor cells through a combination of novel retroviral vector packaging cell lines, and improved vector supernatant production . The new ProPak packaging cell lines produce either murine leukemia virus (MLV) xenotropic (ProPak-X cells) or amphotropic particles (ProPak-A cells), and ProPak-based producer cells were demonstrated to be free of replication-competent retrovirus (RCR) by stringent testing . Vector supernatants from ProPak or existing packaging cell lines producing different pseudotyped particles (amphotropic MLV, xenotropic MLV or gibbon ape leukemia virus) were compared for the ability to transduce clinically relevant human hematopoietic cells . All vector types transduced primary human CD34-positive or CD4-positive cells, regardless of tropism . However, consistently higher transduction of target cells was achieved with ProPak-derived amphotropic vector than with PA317-packaged amphotropic vector . The highest transduction of human hematopoietic progenitor cells was achieved with vector supernatant generated from a coculture of the ProPak-X and ProPak-A cell lines . This ping-pong amplification yielded supernatant containing vector targeted to two distinct receptors present on human cells, and did not result in detectable RCR formation . In addition, we describe conditions for improved vector supernatant production in a packed-bed bioreactor. J Hepatol, 1997 Jun, 26(6), 1379 - 92 In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates; Flendrig LM et al.; BACKGROUND/AIMS: The development of custom-made bioreactors for use as a bioartificial liver (BAL) is considered to be one of the last challenges on the road to successful temporary extracorporeal liver support therapy . We devised a novel bioreactor (patent pending) which allows individual perfusion of high density cultured hepatocytes with low diffusional gradients, thereby more closely resembling the conditions in the intact liver lobuli . METHODS: The bioreactor consists of a spirally wound nonwoven polyester matrix, i.e . a sheet-shaped, three-dimensional framework for hepatocyte immobilization and aggregation, and of integrated hydrophobic hollow-fiber membranes for decentralized oxygen supply and CO2 removal . Medium (plasma in vivo) was perfused through the extrafiber space and therefore in direct hepatocyte contact . Various parameters were assessed over a period of 4 days including galactose elimination, urea synthesis, lidocaine elimination, lactate/pyruvate ratios, amino acid metabolism, pH, the last day being reserved exclusively for determination of protein secretion . RESULTS: Microscopic examination of the hepatocytes revealed cytoarchitectural characteristics as found in vivo . The biochemical performance of the bioreactor remained stable over the investigated period . The urea synthesizing capacity of hepatocytes in the bioreactor was twice that of hepatocytes in monolayer cultures . Flow sensitive magnetic resonance imaging (MRI) revealed that the bioreactor construction ensured medium flow through all parts of the device irrespective of its size . CONCLUSIONS: The novel bioreactor showed encouraging efficiency . The device is easy to manufacture with scale-up to the liver mass required for possible short-term support of patients in hepatic failure. In Vitro Cell Dev Biol Anim, 1997 Jun, 33(6), 459 - 66 Three-dimensional growth patterns of various human tumor cell lines in simulated microgravity of a NASA bioreactor; Ingram M et al.; Growth patterns of a number of human tumor cell lines that from three-dimensional structures of various architectures when cultured without carrier beads in a NASA rotary cell culture system are described and illustrated . The culture system, which was designed to mimic microgravity, maintained cells in suspension under very low-shear stress throughout culture . Spheroid (particulate) production occurred within a few hours after culture was started, and spheroids increased in size by cell division and fusion of small spheroids, usually stabilizing at a spheroid diameter of about 0.5 mm . Architecture of spheroids varied with cell type . Cellular interactions that occurred in spheroids resulted in conformation and shape changes of cells, and some cell lines produced complex, epithelial-like architectures . Expression of the cell adhesion molecules, CD44 and E cadherin, was upregulated in the three-dimensional constructs . Coculture of fibroblast spheroids with PC3 prostate cancer cells induced tenascin expression by the fibroblasts underlying the adherent prostate epithelial cells . Invasion of the fibroblast spheroids by the malignant epithelium was also demonstrated. Appl Environ Microbiol, 1997 Jun, 63(6), 2366 - 71 Silencing MIG1 in Saccharomyces cerevisiae: effects of antisense MIG1 expression and MIG1 gene disruption; Olsson L et al.; Silencing of MIG1, a transcription factor imposing carbon catabolite repression on invertase, was attempted, either by disrupting the gene or by expressing antisense copies of the gene . The performance of the recombinant strains in bioreactor batch cultivations on sucrose, in the presence of glucose, was compared with that of the wild-type strain under the same conditions . In the delta migI strain, the rate of sucrose utilization was independent (10 mmol/g/h) of the glucose concentration . During the cultivations with the wild-type strain and the antisense strains, two distinct phases were observed . The rates of sucrose hydrolysis were < 1 mmol/g/h and 9 to 10 mmol/g/h in the first and second phases, respectively . Entry into the second cultivation phase was characterized by a decline in glucose concentration below 12 mmol/liter . As expected, disruption of MIG1 resulted in a relief of glucose repression . However, silencing of MIG1 expression was not achieved by expressing antisense MIG1, even though antisense MIG1 RNA was sufficiently stable to be detected . In the wild-type and delta migI strains, the specific growth rate was 0.32 to 0.33 h-1, whereas it was lower in the antisense strains, 0.25 to 0.30 h-1. J Biotechnol, 1997 May 23, 55(1), 31 - 41 Plasmid instability kinetics in continuous culture of a recombinant Saccharomyces cerevisiae in airlift bioreactor; Zhang Z et al.; Plasmid instability of a recombinant Saccharomyces cerevisiae C468/pGAC9 (ATCC 20690) was examined during continuous culture in a nonselective medium in an airlift bioreactor . The recombinant strain contained a 2-micron based shuttle vector pGAC9 and expresses Aspergillus awatnori glucoamylase gene under the control of the yeast enolase I (ENO1) promoter . The changes in the fraction of plasmid-bearing cells and glucoamylase activity followed first-order kinetics . Expressed as a function of time, the decay rates of both the plasmid-bearing cell fraction and glucoamylase expression increased with increasing dilution rates . If expressed as a function of cell generations, the decay rates were nearly constant over the dilution rates tested . The results indicated that the growth rate difference between plasmid-bearing and plasmid-free cells was negligible . This was probably due to the low copy number of the 2-micron based yeast shuttle vector . Thus the contribution of preferential growth to apparent plasmid instability was negligible . A novel numerical method is proposed to evaluate the parameters related to plasmid stability . The estimated values of probability of plasmid loss (P = 0.0499) were nearly constant at different dilution rates . No significant effect of growth rates on plasmid instability was observed . The proposed kinetics agreed well with experimental observations. Curr Opin Hematol, 1997 May, 4(3), 157 - 62 Use of stem cell factor to mobilize hematopoietic progenitors; Bearman SI; Stem cell factor (SCF) is a multipotent growth factor that plays a role in the growth and development of hematopoietic cells, spermatocytes, melanocytes, and mast cells . SCF alone has little direct stimulatory activity on hematopoietic progenitors but acts synergistically with other colony-stimulating factors to potentiate their effects on colony growth . SCF also potentiates the mobilization of hematopoietic progenitor cells into the peripheral blood in response to granulocyte colony-stimulating factor (G-CSF), with or without chemotherapy . The combination of SCF plus G-CSF appears to be a particularly effective mobilization regimen for patients who have been heavily pretreated . Whether engraftment of peripheral blood progenitor cells mobilized by SCF plus G-CSF is superior to that of cells mobilized by G-CSF alone remains unclear . SCF appears to be a necessary requirement for ex vivo expansion of hematopoietic progenitors in both liquid and bioreactor culture systems and will be an important component of future cellular therapies, such as expansion of placental cord blood progenitors and retroviral-mediated gene transfer into hematopoietic target cells. In Vitro Cell Dev Biol Anim, 1997 May, 33(5), 386 - 91 Skeletal muscle satellite cells cultured in simulated microgravity; Molnar G et al.; Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells . Satellite cells retain the capacity to proliferate and differentiate in vitro and, therefore, provide a useful model to study postnatal muscle development . Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish . Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo . Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development . Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75% satellite cells . Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV . Plating efficiency, proliferation, and glucose utilization were compared between 2-D culture and 3-D HARV culture . Plating efficiency (cells attached divided by cells plated x 100) was similar between the two culture systems . Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and nonsatellite cells . Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability because glucose levels in medium from HARV and 2-D cell culture were similar . Morphologically, microcarrier beads within the HARV were joined together by cells into 3-D aggregates composed of greater than 10 beads/aggregate . Aggregation of beads did not occur in the absence of cells . Myotubes were often seen on individual beads or spanning the surface of two beads . In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a 3-D level of organization that could provide a more suitable model to study postnatal muscle development than is currently available with standard culture methods. In Vitro Cell Dev Biol Anim, 1997 May, 33(5), 381 - 5 Microgravity tissue engineering; Freed LE et al.; Tissue engineering studies were done using isolated cells, three-dimensional polymer scaffolds, and rotating bioreactors operated under conditions of simulated microgravity . In particular, vessel rotation speed was adjusted such that 10 mm diameter x 2 mm thick cell-polymer constructs were cultivated in a state of continuous free-fall . Feasibility was demonstrated for two different cell types: cartilage and heart . Conditions of simulated microgravity promoted the formation of cartilaginous constructs consisting of round cells, collagen and glycosaminoglycan (GAG), and cardiac tissue constructs consisting of elongated cells that contracted spontaneously and synchronously . Potential advantages of using a simulated microgravity environment for tissue engineering were demonstrated by comparing the compositions of cartilaginous constructs grown under four different in vitro culture conditions: simulated microgravity in rotating bioreactors, solid body rotation in rotating bioreactors, turbulent mixing in spinner flasks, and orbital mixing in petri dishes . Constructs grown in simulated microgravity contained the highest fractions of total regenerated tissue (as a percent of construct dry weight) and of GAG, the component required for cartilage to withstand compressive force. In Vitro Cell Dev Biol Anim, 1997 May, 33(5), 352 - 7 Induction of carcinoembryonic antigen expression in a three-dimensional culture system; Jessup JM et al.; MIP-101 is a poorly differentiated human colon carcinoma cell line established from ascites that produces minimal amounts of carcinoembryonic antigen (CEA), a 180 kDa glycoprotein tumor marker, and nonspecific cross-reacting antigen (NCA), a related protein that has 50 and 90 kDa isoforms, in monolayer culture . However, MIP-101 produces CEA when implanted into the peritoneum of nude mice but not when implanted into subcutaneous tissue . We tested whether three-dimensional (3D) growth was a sufficient stimulus to produce CEA and NCA 50/90 in MIP-101 cells, because cells grow in 3D in vivo rather than in two-dimensions (2D) as occurs in monolayer cultures . To do this, MIP-101 cells were cultured on microcarrier beads in 3D cultures, either in static cultures as nonadherent aggregates or under dynamic conditions in a NASA-designed low shear stress bioreactor . MIP-101 cells proliferated well under all three conditions and increased CEA and NCA production three- to four-fold when grown in 3D cultures compared to MIP-101 cells growing logarithmically in monolayers . These results suggest that 3D growth in vitro simulates tumor function in vivo and that 3D growth by itself may enhance production of molecules that are associated with the metastatic process. In Vitro Cell Dev Biol Anim, 1997 May, 33(5), 337 - 43 Neonatal rat heart cells cultured in simulated microgravity; Akins RE et al.; In vitro characteristics of cardiac cells cultured in simulated microgravity are reported . Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane . Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types . Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system . The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue . We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity . The NASA-designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension . HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques . Both systems were uniformly inoculated and medium exchanged at standard intervals . Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization . Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type . In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures . Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organization of cardiac cells in vitro. In Vitro Cell Dev Biol Anim, 1997 May, 33(5), 332 - 6 Cultivation of fall armyworm ovary cells in simulated microgravity; Francis KM et al.; A methodology is presented to culture Fall Armyworm Ovary cells in simulated micrograviy using a novel bioreactor developed by NASA, the High-Aspect Ratio Vessel . In this vessel, the growth and metabolic profile for these insect cells were profoundly different than those obtained in shaker-flask culture . Specifically, stationary phase in the NASA vessel was extended from 24 h to at least 7 d while cell concentration and viability remained in excess of 1 x 10(7) viable cells/ml and 90%, respectively . Measurements of glucose utilization, lactate production, ammonia production, and pH change indicate that simulated microgravity had a twofold effect on cell metabolism . Fewer nutrients were consumed and fewer wastes were produced in stationary phase by as much as a factor of 4 over that achieved in shaker culture . Those nutrients that were consumed in the NASA vessel were directed along different metabolic pathways as evidenced by an extreme shift in glucose utilization from consumption to production in lag phase and a decrease in yield coefficients by one half in stationary phase . These changes reflect a reduction in hydrodynamic forces from over 1 dyne/cm2 in shaker culture to under 0.5 dyne/cm2 in the NASA vessel . These results suggest that cultivation of insect cells in simulated microgravity may reduce production costs of cell-derived biologicals by extending production time and reducing medium requirements. Neth J Med, 1997 May, 50(5), 228 - 32 Gastro-entero-hepatology in the next millennium; Tytgat GN; The scope of gastroenterology and hepatology now and its development into the next century are great and expanding . Only some of the many exciting improvements which are expected during the next few years can be discussed here . Progress in the microbial aetiology and novel targeted treatments for inflammatory bowel disease (IBD) will be paralleled by better understanding of functional upper and lower gut disorders, their neural control and neuropharmacological treatment . Technical developments in the pipeline include improvements in endoscopic and endosonographic equipment and techniques, including MR- or CT-based 'virtual colonoscopy' to obviate many invasive diagnostic colonoscopies, and the remarkable self-propelled endoscope, which will worm its way to regions of interest in the bowel . In hepatology, transplantation, and vaccination for hepatitides will increase, and improved treatment of acute liver failure may involve bioreactors or cryopreserved human hepatocytes . Gastrointestinal oncology will progress through more extensive surgery, gene therapy and techniques such as mucosal resection . A target force of about 150 trained gastroenterologist/hepatologists will be needed in the Netherlands--one for every 100,000 of the population. Biochem Biophys Res Commun, 1997 Apr 7, 233(1), 238 - 43 A radiobiological probe for simultaneous NMR spectroscopy and 192Ir gamma irradiation of Saccharomyces cerevisiae; Magness JE et al.; A novel nuclear magnetic resonance (NMR) spectroscopy probe was designed and constructed for the study of transient metabolic changes in cellular systems during exposure to ionizing radiation . The probe incorporated a bioreactor, a radiation source, and a radiofrequency detection circuit tunable between 100 and 300 MHz for in vivo NMR spectroscopy of 23Na, 13C, and 31P at 11.7 Tesla . The bioreactor system allowed perfusion, oxygenation, and temperature control of cultured cells during irradiation and while performing simultaneous spectroscopic experiments . The concentric design of the bioreactor allowed for the insertion of a 192Ir gamma ray source (E(gamma) = 370 keV) to allow irradiation of the bioreactor system during the acquisition of NMR spectra . Initial results of 31P spectra obtained during simultaneous gamma irradiation of Saccharomyces cerevisiae at approximately 8 Gy/hr show rapid decreases in adenosine triphosphate (ATP) and polyphosphate at the onset of irradiation followed by a slow recovery of polyphosphate. J Biotechnol, 1997 Apr 4, 54(1), 1 - 14 Regulation of a continuous yeast bioreactor near the critical dilution rate using a productostat; Andersen MY et al.; Regulation of a continuous bioreactor with Saccharomyces cerevisiae is investigated . A number of different sensors are evaluated for this purpose and the process dynamics is investigated around the critical dilution rate . A sensor for reducing gas concentration in exhaust gases is selected for regulating the substrate flow rate . Closed loop identification experiments are carried out to enable identification of the process dynamics near the critical diluton rate . Due to the time-varying nature of this process an adaptive regulator seems to be a promising tool for providing good regulatory and setpoint tracking performance . A simple third order model is used for a model based control design with a Linear Quadratic (LQ)-regulator . The LQ-regulator performs well experimentally, both in an adaptive version where the model parameters are updated on-line, and in a non-adaptive version . During the test the process is exposed to a large disturbance in substrate feed concentration and to a small setpoint disturbance . The proposed regulator is a practical realisation of a productostat where the product in this case is an undesired primary metabolite . Thus, this paper demonstrates a more general principle of utilizing metabolic overflow metabolism for directing fluxes through a desired metabolic pathway . This principle is applicable in the presented form, if a (by-)product can be measured on-line. Curr Opin Biotechnol, 1997 Apr 1, 8(2), 169 - 74 Partitioning bioreactors; Daugulis AJ; A very wide range of methods has recently been employed to selectively partition fermentation products, substrates, or other metabolites to achieve an overall improvement in bioprocess performance . Techniques ranging from extraction and gas stripping to electrokinetic methods have been used to favorably influence the microenvironment of cells being cultivated in bioreactors . Virtually all types of cells, including animal cells, have benefitted by such selective partitioning and very high value fermentation products, such as monoclonal antibodies and secondary metabolites, have been produced using this process concept. Curr Opin Biotechnol, 1997 Apr 1, 8(2), 165 - 8 Innovative bioreactors; Deshusses MA et al.; Recent papers have described new bioreactor designs . Most innovations addressed either oxygen transfer, shear induced by stirring, control of water activity in organic phase systems or waste biotreatment . Innovations made during the past year were reported in mainly three areas: bioreactor designs for increases in oxygen transfer and decreases in shear stress; bioreactors for two-phase reactions with water activity control; and environmental bioreactors. Biotechnol Prog, 1997 Mar-Apr, 13(2), 185 - 94 Mist deposition onto hairy root cultures: aerosol modeling and experiments; Wyslouzil BE et al.; We analyzed the applicability of the standard models for aerosol deposition in randomly packed fibrous filter beds to mist deposition across a bed of hairy roots in the nutrient mist bioreactor . Although the assumptions inherent in the models are met on a local level, the overall structure of the root bed introduces some uncertainty into the correct choice of root packing fraction and gas velocity required by the model . For reasonable parameter values, the minimum in the deposition efficiency curves is close to the peak in the mist number and mass distributions, and good penetration of the root bed is possible . We then measured the deposition of mist across a packed bed of Artemisia annua transformed roots as a function of droplet size, bed length, and gas flow rate at a root packing fraction alpha = 0.5 . We compared the experimental measurements with the predictions of the aerosol deposition model and found good agreement between the measured and predicted values for the diameter where the deposition efficiency across the bed is 50%, D0.5 . Agreement between the model and the experiments broke down when the flow rate was increased to the point where the creeping flow assumptions were no longer valid. Biotechnol Prog, 1997 Mar-Apr, 13(2), 151 - 5 High-level expression of a heterologous gene in Escherichia coli in response to carbon-nitrogen source and C/N ratio in a batch bioreactor; Bhattacharya SK et al.; Overexpression of the target gene in a recombinant strain of Escherichia coli has been analyzed in view of changes in the carbon-nitrogen source and as a function of C/N ratio . The rate of target gene expression varied over a range of 200%, and the yield coefficient for the target gene product changed over 400% with change in carbon source at 10 gL-1 in M9 medium . With variation in nitrogen source, however, only marginal changes (10-15%) occurred in these parameters of overexpression . Higher concentration, for example 2.5 g L-1, of any nitrogen source adversely affected heterologous expression as rate of target gene expression declined in the range 35%-50% and the yield coefficient of foreign protein decreased between 45% and 60% . A C/N ratio of 15 was found to be optimum for both parameters for overexpression and host specific parameters such as specific growth rate and observed yield coefficient for cellular growth . An analysis with respect to temperature and pH revealed that host and expression parameters were quite susceptible to changes in these factors. Appl Biochem Biotechnol, 1997 Feb-Mar, 62(2-3), 291 - 302 The culture of chicken embryo fibroblast cells on microcarriers to produce infectious bursal disease virus; Zhang L et al.; The cultures of chicken embryo fibroblast (CEF) cells in flasks, spinner bottles, and bioreactors were studied . The growth and metabolism characteristics of CEF cells and the feasibility of the CEF cell culture in bioreactor were investigated . The plating process of the CEF cells on GT-2 microcarriers in spinner bottles was studied, and a plating kinetic model was presented . The culture of CEF cells in 1.5 L CelliGen bioreactor to produce infectious bursal disease virus (IBDV) had met success . Whereas the additive microcarriers were fed during the culture, the cell density was increased 10 times as against seed cells adhering to microcarriers and the virus titer was as high as 7.5 . All the aforementioned experimental results have laid the foundation for high density culture of CEF cells and process scale-up. Int J Artif Organs, 1997 Feb, 20(2), 119 - 24 Comparative evaluation of different membranes for the construction of an artificial liver support system; Qiang S et al.; During the past decades, many technological improvements have been made in the construction of extracorporeal liver support systems . Among these achievements, membranes of artificial capillary system, used as substrates of hepatocyte growth, aroused our interest in their application for the construction of bioreactors . The present paper studied the comparison of hepatocyte growth and function on six different membranes . Four of them are cellulose based membranes, Cuprophan, Hemophan, Cellulose acetate, and Bioflux; two are synthetic polymer SPAN and Polysulphone . Human hepatoma cell line SMMC-7721, with moderately differentiated hepatocyte-specific functions, was inoculated into the hollow fiber cartridges . These cells were allowed to attach and to grow over these membranes . It was found that there existed differences in hepatocyte immobilization and growth among these membranes . They influenced the growth and functions of hepatoma cells in vitro to some extent . These results show that membrane is an important factor in the construction of capillary membrane bioreactors for artificial liver support. Trends Biotechnol, 1997 Feb, 15(2), 45 - 50 Plants as bioreactors for biopharmaceuticals: regulatory considerations; Miele L; Plants are one of several novel hosts that can be used for the production of recombinant biopharmaceuticals such as cytokines, hormones, monoclonal antibodies, enzymes and vaccines . The novelty of this technology and its wide range of potential applications require an assessment of possible regulatory concerns in the clinical development of plant-derived biopharmaceuticals . General principles extrapolated from experience gained with biotechnology products from other sources can serve as a foundation to develop scientifically sound strategies for the large-scale production and clinical development of safe and effective biopharmaceuticals in plant hosts. J Biomed Mater Res, 1997 Feb, 34(2), 211 - 20 Evaluation of matrix scaffolds for tissue engineering of articular cartilage grafts; Grande DA et al.; Injury to articular cartilage predisposes that joint to further degeneration and eventually osteoarthritis . Recent studies have demonstrated the feasibility of using chondrocytes together with different biomaterial carriers as grafts for the repair of cartilage defects . The following study was undertaken to determine the effect of a variety of these materials on chondrocyte growth and extracellular matrix synthesis . We cultured chondrocytes on several commonly used materials and compared their rates of synthesis of proteoglycan and collagen . Additionally, we evaluated them in a closed culture recirculating system on these materials and compared them with standard culture techniques . This was done to see whether such a bioreactor-type system can be used to enhance the quality of in vitro reconstructed tissues . Our results demonstrated marked variability with respect to how chondrocytes responded to culture on the various materials . Bioabsorbable polymers such as polyglycolic acid (PGA)--enhanced proteoglycan synthesis, whereas collagen matrices stimulated synthesis of collagen . The use of the closed culture system, in general, improved the rates of synthesis of collagen and proteoglycan on the different material scaffolds . Exceptions were collagen synthesis on collagen matrices: use of the closed culture system did not enhance the rate of synthesis . Rates of proteoglycan synthesis on PGA scaffold initially was higher in the closed culture system but did not sustain a difference over the entire course of the 3-week culture period . This study demonstrates the importance of carrier material for the purpose of cartilage tissue reconstruction in vitro. J Immunol Methods, 1997 Jan 15, 200(1-2), 39 - 46 Determination of anti-HBsAg IgM monoclonal antibodies in cell culture media by perfusion immunoassay; Brackett JM et al.; A rapid, specific, perfusion immunoassay for active anti-HBsAg monoclonal IgM is described . The immunoassay requires less than 3.5 min per sample . The precision was found to be 3.6% at an IgM concentration of 17 microg/ml . A detection limit of 1 microg/ml IgM in culture media was determined . Assay results were found to correlate very well with standard size exclusion chromatography and radial immunodiffusion techniques . This perfusion immunoassay was demonstrated to be useful for determining anti-HBsAg IgM in complex matrices such as cell culture media . The utility of the immunoassay for monitoring production of anti-HBsAg IgM in a perfusion bioreactor is demonstrated. Chin J Biotechnol, 1997, 13(1), 51 - 8 Engineering factors affecting the granulation of sludge in the UASB reactor; Guo Y et al.; The influence of inoculum sludge involving different microbial populations, different modes of stream flow and different flow rates in a bioreactor on the granulation of sludge was studied . In the granulation process there were four periods: formation of microbial floccus, formation of subcore, growth of subcore, and maturity of granules . The production of microfloccus could be attributed to acid-forming bacteria . The hydrodynamic momentum transfer caused by moving phases and the hydraulic shear effect are the crucial factors affecting the formation of the subcore . For this a new concept-the lowest limited flow rate-is proposed, i.e., the lowest flow rate to form an expanded sludge bed . A sufficient feed rate (higher than the lowest flow rate), an appropriate sludge load, and an equal distribution of feed, as well as a suitable alkalinity in the medium are the essential factors for the scaling-up and process-control of the UASB reactor. Immunogenetics, 1997, 45(6), 379 - 85 Large-scale production of class I bound peptides: assigning a signature to HLA-B*1501; Prilliman K et al.; A peptide-based vaccine must be bound and presented by major histocompatibility complex class I molecules to elicit a CD8(+) T-cell response . Because class I HLA molecules are highly polymorphic, it has yet to be established how well a vaccine peptide that stimulates one individual's CD8(+) cytotoxic T lymphocytes will be presented by a second individual's different class I molecules . Therefore, to facilitate precise comparisons of class I peptide binding overlaps, we uniquely combined hollow-fiber bioreactors and mass spectrometry to assign precise peptide binding signatures to individual class I HLA molecules . In applying this strategy to HLA-B*1501, we isolated milligram quantities of B*1501-bound peptides and mapped them using mass spectrometry . Repeated analyses consistently assign the same peptide binding signature to B*1501; the degree of peptide binding overlap between any two class I molecules can thus be determined through comparison of their peptide signatures. J Ind Microbiol Biotechnol, 1997 Jan, 18(1), 22 - 5 Secondary metabolism in simulated microgravity: beta-lactam production by Streptomyces clavuligerus; Fang A et al.; Rotating bioreactors designed at NASA's Johnson Space Center were used to simulate a microgravity environment in which to study secondary metabolism . The system examined was beta-lactam antibiotic production by Streptomyces clavuligerus . Both growth and beta-lactam production occurred in simulated microgravity . Stimulatory effects of phosphate and L-lysine, previously detected in normal gravity, also occurred in simulated microgravity . The degree of beta-lactam antibiotic production was markedly inhibited by simulated microgravity. Adv Space Biol Med, 1997, 6, 255 - 74 Bioregeneration with maltose excreting Chlorella: system concept, technological development, and experiments; Wolf L; ESA has been studying a small-scale bioregenerative system to support long-term biological experiments on-board spacecraft with oxygen, water and food . Core component of this system is a special photo-bioreactor in which a maltose-producing strain of the green micro alga Chlorella is cultivated . A number of auxiliary system components have been developed and are functioning on the ground according to the design specifications, among them a gas/liquid phase separator operating at the same time as a low shear-stress pneumatic pump, a dehumidifier, a maltose separator, and a liquid transfer system . All components have been designed so that--in principle--they will operate in weightlessness, though this has so far only been verified for the gas/liquid separator . The bioreactor and some of the auxiliary components have been integrated in a prototype system, which has been subjected to preliminary testing . The prototype has been sterilized successfully by autoclaving, except for the liquid transfer unit which is disinfected with isopropyl alcohol . Chlorella 241.80 has been cultured several times under controlled conditions for up to 8 weeks . Algal growth to a biomass concentration of 9 g.l-1 dry weight and maltose production to a concentration of 17 g.l-1 have been achieved . The low shear-stress pneumatic pump works reliably without the mechanical cell damage produced by other types of pumps . Contamination of the algal cultures by other micro-organisms has been avoided in most of the experiment runs . The maximum oxygen production rate observed was 2 ml.min-1, when the culture was aerated with air +0.5% CO2 . This production rate is well below the CO2 gas transfer rate of 5 ml.min-1 under these conditions . It can probably be doubled by increasing the maximum light intensity of the illumination unit (currently 300 micro E.m-2S-1) . In a preliminary closed gas loop experiment with Periplaneta as consumer, the possibility of controlling the Chlorella culture so as to match the needs of the consumer colony has been established . A maltose excreting Chlorella strain has been selected as the photosynthetic producer, because the technique for automatic culturing of this organism and harvesting its products was expected to be much less complex than that required for culturing higher plants . Although the prototype system developed in our laboratory has reached a high level of sophistication, there remain still a number of technical and biological problems to be solved before the feasibility of this concept is definitely demonstrated . The major problem is maintaining sterility, and eventually automatic cleaning and resterilization when contamination occurs during operation . The culture medium, which contains minerals, cell fragments and considerable amounts of sugars, is an ideal substrate for many other microorganisms . Another problem is long term operation . The prototype system contains many tubes and ducts which are perfused with culture medium . These may clog, which may lead to loss of sensor information essential for controlling the culture . Even when we succeed in demonstrating the feasibility of this concept, it will be a difficult task to demonstrate convincingly that the expected advantages of a bioregenerative system can outweigh the simplicity and reliability of a non-regenerative stored resource system in terms of volume, mass and amount of consumables required over the operational time. Microbiology, 1997 Jan, 143 ( Pt 1), 203 - 18 Flux distributions in anaerobic, glucose-limited continuous cultures of Saccharomyces cerevisiae; Nissen TL et al.; A stoichiometric model describing the anaerobic metabolism of Saccharomyces cerevisiae during growth on a defined medium was derived . The model was used to calculate intracellular fluxes based on measurements of the uptake of substrates from the medium, the secretion of products from the cells, and of the rate of biomass formation . Furthermore, measurements of the biomass composition and of the activity of key enzymes were used in the calculations . The stoichiometric network consists of 37 pathway reactions involving 43 compounds of which 13 were measured (acetate, CO2, ethanol, glucose, glycerol, NH4+, pyruvate, succinate, carbohydrates, DNA, lipids, proteins and RNA) . The model was used to calculate the production rates of malate and fumarate and the ethanol measurement was used to validate the model . All rate measurements were performed on glucose-limited continuous cultures in a high-performance bioreactor . Carbon balances closed within 98% . The calculations comprised flux distributions at specific growth rates of 0.10 and 0.30 h-1 . The fluxes through reactions located around important branch points of the metabolism were compared, i.e . the split between the pentose phosphate and the Embden-Meyerhoff-Parnas pathways . Also the model was used to show the probable existence of a redox shunt across the inner mitochondrial membrane consisting of the reactions catalysed by the mitochondrial and the cytosolic alcohol dehydrogenase . Finally it was concluded that cytosolic isocitrate dehydrogenase is probably not present during growth on glucose . The importance of basing the flux analysis on accurate measurements was demonstrated through a sensitivity analysis . It was found that the accuracy of the measurements of CO2, ethanol, glucose, glycerol and protein was critical for the correct calculation of the flux distribution. Appl Environ Microbiol, 1997 Jan, 63(1), 122 - 7 Stable expression of pertussis toxin in Bordetella bronchiseptica under the control of a tightly regulated promoter; Suarez A et al.; Pertussis toxin (PT) is an essential component of accellular vaccines against whooping cough . However, the industrial production of PT from Bordetella pertussis is impaired by slow growth and poor yields . To overcome these problems, we have constructed a minitransposon containing the tox operon under the control of a tightly regulated promoter responsive to an aromatic inducer . The expression cassettes have been integrated into the chromosome of Bordetella bronchiseptica 5376 and ATCC 10580 bvg . Five recombinant clones containing the tox operon under the control of the Psal promoter, which is activated by the product of nahR, were further characterized . The recombinant clones expressed PT after only 3 h of induction with sodium salicylate at levels similar to those of B . pertussis grown for 24 h . The stability of the engineered phenotype was 100% after 72 h of growth without selective pressure . The growth pattern was not modified either under noninducing conditions or in the presence of the inducer at low concentrations, suggesting that strain performance would not be affected in bioreactors when uncoupled from gene expression . Recombinant PT, which was localized mainly in the periplasm, was purified by affinity chromatography . The recombinant protein was immunologically indistinguishable from wild-type PT and retained its biological activity as determined by the CHO cell-clustering test . These recombinant clones appear to be useful tools for the cost-effective production of PT under conditions of improved biosafety, as demonstrated by the inducible expression of PT uncoupled from the bacterial biomass in