ROLE OF BIOREACTOR IN BIOPROCESS ENGG.-03

Bioseparation, 2001, 10(1-3), 57 - 63Capture of proteins from mammalian cells in pilot scale using different STREAMLINE adsorbents; Lutkemeyer D et al.; This presentation compares three different expanded bed matrices . STREAMLINE rProtein A, STREAMLINE SP-XL and STREAMLINE Chelating were monitored in respect to their ability to clarify the broth, to concentrate and to purify the distinct target protein . The capture of a mouse IgG1 and a recombinant prothrombin (PT) was carried out in pilot scale using a 100-l bioreactor and STREAMLINE 100 and 200 columns, respectively . The robustness of the process was also estimated monitoring the expansion behaviour and the cell and debris concentrations during the load and in the eluat . In all cases the capture of the target proteins was comparable to conventional chromatographic systems . The purification success was mainly dependent on the selectivity of the ligand used . The affinity process resulted in a highly purified product . The ion exchanger and chelating material mainly concentrated the product . In all three cases 100 l of cell broth were successfully processed in one run . The robustness of the ion exchanger process was poor, because of strong cell matrix interaction . However, for the chelating and especially for the affinity matrix a highly reproducible process was obtained.Biotechnol Bioeng, 2002 Feb 15, 77(4), 455 - 61Overexpression of alcohol dehydrogenase or pyruvate decarboxylase improves growth of hairy roots at reduced oxygen concentrations; Shiao TL et al.; Overexpression of Arabidopsis thaliana genes for the fermentation enzymes, alcohol dehydrogenase and pyruvate decarboxylase, improved the tolerance of A . thaliana hairy roots to low oxygen conditions . Whereas the specific growth rate of untransformed hairy roots in shake flasks and in a multiple-tube recirculation bioreactor declined significantly with decreasing oxygen tension down to 25% air saturation, growth of the transformant root lines was maintained at rates similar to those achieved with full aeration . This work demonstrates that altering the expression of selected genes involved in anaerobic metabolism can alleviate the problems of oxygen deficiency in hairy root cultures caused by poor mixing and mass transfer conditions . Biotechnol Bioeng, 2002 Feb 15, 77(4), 381 - 93Validation of a model for process development and scale-up of packed-bed solid-state bioreactors; Weber FJ et al.; We have validated our previously described model for scale-up of packed-bed solid-state fermenters (Weber et al., 1999) with experiments in an adiabatic 15-dm(3) packed-bed reactor, using the fungi Coniothyrium minitans and Aspergillus oryzae . Effects of temperature on respiration, growth, and sporulation of the biocontrol fungus C . minitans on hemp impregnated with a liquid medium were determined in independent experiments, and the first two effects were translated into a kinetic model, which was incorporated in the material and energy balances of the packed-bed model . Predicted temperatures corresponded well with experimental results . As predicted, large amounts of water were lost due to evaporative cooling . With hemp as support no shrinkage was observed, and temperatures could be adequately controlled, both with C . minitans and A . oryzae . In experiments with grains, strong shrinkage of the grains was expected and observed . Nevertheless, cultivation of C . minitans on oats succeeded because this fungus did not form a tight hyphal network between the grains . However, cultivation of A . oryzae failed because shrinkage combined with the strong hyphal network formed by this fungus resulted in channeling, local overheating of the bed, and very inhomogeneous growth of the fungus . For cultivation of C . minitans on oats and for cultivation of A . oryzae on wheat and hemp, no kinetic models were available . Nevertheless, the enthalpy and water balances gave accurate temperature predictions when online measurements of oxygen consumption were used as input . The current model can be improved by incorporation of (1) gas-solids water and heat transfer kinetics to account for deviations from equilibrium observed with fast-growing fungi such as A . oryzae, and (2) the dynamic response of the fungus to changes in temperature, which were neglected in the isothermal kinetic experiments . J Endocrinol, 2002 Jan, 172(1), 199 - 210Production and purification of recombinant human inhibin and activin; Pangas SA et al.; Inhibin and activin are protein hormones with diverse physiological roles including the regulation of pituitary FSH secretion . Like other members of the transforming growth factor-beta gene family, they undergo processing from larger precursor molecules as well as assembly into functional dimers . Isolation of inhibin and activin from natural sources can only produce limited quantities of bioactive protein . To purify large-scale quantities of recombinant human inhibin and activin, we have utilized stably transfected cell lines in self-contained bioreactors to produce protein . These cells produce approximately 200 microg/ml per day total recombinant human inhibin . Conditioned cell media can be purified through column chromatography resulting in dimeric mature 32-34 kDa inhibin A and 28 kDa activin A . The purified recombinant proteins maintain their biological activity as measured by traditional in vitro assays including the regulation of FSH in rat anterior pituitary cultures and the regulation of promoter activity of the activin-responsive promoter p3TP-luc in tissue culture cells . These proteins will be valuable for future analysis of inhibin and activin function and have been distributed to the US National Hormone and Peptide Program.Med Clin (Barc), 2001 Dec 15, 117(20), 781 - 4{Bioartificial liver support for acute liver failure . First case treated in Spain}; Salmeron JM et al.; BACKGROUND: Research aimed at developing artificial liver support systems has experienced a notable increase in the last decade . Hybrid systems including bioreactors containing hepatocytes which are perfused by liver failure patients blood or plasma have been deviced for the first time . The purpose of such a strategy is to substitute, at least in part, the impaired hepatic function thus improving the prognosis of patients with severe acute or chronic liver diseases . CASE REPORT: In the present paper, we report the first such a case treated in Spain in the context of a controlled, randomized, multicenter international study aimed at investigating the usefulness and safety of a bioartificial liver support system based on cryopreserved porcine hepatocytes in patients with acute liver failure or having a non-functioning primary graft after liver transplantation . RESULTS: In this first experience, two sessions of treatment could be completed before a patient with acute liver failure underwent a successful emergency liver transplantation . After more than two years of follow-up, the patient is in her normal life activities and she has not presented any adverse event related to the bioartificial liver support therapy so far . CONCLUSION: Bioartificial liver support systems are starting to be available for use in clinical practice . Yet it is mandatory to establish their safety and efficacy before a widespread recommendation.J Orthop Res, 2001 Nov, 19(6), 1089 - 97Integration of engineered cartilage; Obradovic B et al.; The structure and function of cartilaginous constructs, engineered in vitro using bovine articular chondrocytes, biodegradable scaffolds and bioreactors, can be modulated by the conditions and duration of tissue cultivation . We hypothesized that the integrative properties of engineered cartilage depend on developmental stage of the construct and the extracellular matrix content of adjacent cartilage, and that some aspects of integration can be studied under controlled in vitro conditions . Disc-shaped constructs (cultured for 5+/-1 days or 5+/-1 weeks) or explants (untreated or trypsin treated cartilage) were sutured into ring-shaped explants (untreated or trypsin treated cartilage) to form composites that were cultured for an additional 1-8 weeks in bioreactors and evaluated biochemically, histologically and mechanically (compressive stiffness of the central disk, adhesive strength of the integration interface) . Immature constructs had poorer mechanical properties but integrated better than either more mature constructs or cartilage explants . Integration of immature constructs involved cell proliferation and the progressive formation of cartilaginous tissue, in contrast to the integration of more mature constructs or native cartilage which involved only the secretion of extracellular matrix components. h, h. Integration patterns correlated with the adhesive strength of the disc-ring interface, which was markedly higher for immature constructs than for either more mature constructs or cartilage explants . Trypsin treatment of the adjacent cartilage further enhanced the integration of immature constructs.Zhonghua Kou Qiang Yi Xue Za Zhi, 2000 Jul, 35(4), 254 - 5{A quick way in isolation and amplification of mandibular condylar cartilage cell in vitro}; Jiao Y et al.; OBJECTIVE: To establish a quick way in acquiring well differentiated mandibular condylar cartilage (MCC) cells with high viability in large scale . METHODS: Japan white rabbit MCC cells were harvested by enzymatic method . They were grown in a modified bioreactor culture system, which contained the cytodex-3 micro-carriers in the culture medium . Kinetic growth of MCC cells on DEAE-dextran micro-carrier was observed under phase contrast microscope and environmental scanning microscope respectively . RESULTS: MCC cells attached rapidly to the surface of micro-carriers, but their spreading was slow . A quick growth of these cells was observed when they fully spread onto the micro-carrier . The number of MCC cells increased 16.2 times compared with that of plating . CONCLUSIONS: Micro-carrier culture of MCC cells can yield a large quantity of cells within a short period of time that will be of benefit in banking MCC cells for reconstruction of impaired cartilage.Cancer Biother Radiopharm, 2001 Oct, 16(5), 381 - 90Characterization of human tumor-infiltrating lymphocytes expanded in hollow-fiber bioreactors for immunotherapy of cancer; Malone CC et al.; We attempted to grow tumor-infiltrating lymphocytes (TIL) from 34 fresh tumors of eight different histologies using flasks for the initiation phase and hollow fiber bioreactors to expand TIL to therapeutic numbers . Overall success rate was 76% (26/34) including melanoma (9/14, 64%) and renal cell carcinoma (11/11, 100%) . The mean number of days required to reach successful initiation (1 x 10(9) TIL) for all tumor types was 29 +/- 16 days (mean +/- S.D.) . Therapeutic doses of TIL required an average of 88 +/- 23 days (initiation plus expansion) with an average TIL number of 3.2 x 10(10) +/- 2.8 x 10(10) . TIL phenotype was predominantly CD4+ in 53% (16/30) and CD8+ in 47% (14/30), renal cell carcinoma samples accounted for 12/14 of the predominantly CD8+ TIL . Cells bearing the natural killer (NK) phenotype represented only 0-7% of TIL while LAK phenotype represented 0-68% (mean 11 +/- 15%); LAK was the predominant phenotype in one patient with kidney cancer . Cytotoxicity tests showed consistent NK and LAK activity in addition to cytolysis of autologous tumor . Autologous tumor cell restricted cytolysis was noted for three TIL cultures . The overall success rate and characteristics of TIL were similar to our results with TIL expanded in semi-permeable plastic bags . This work confirms that hollow-fiber bioreactors are a suitable alternative to semi-permeable bags and roller bottle systems for the expansion of human TIL for therapeutic use in cancer patients.FASEB J, 2002 Feb, 16(2), 270 - 2 Epub 2001 Dec 28.Cell differentiation by mechanical stress; Altman GH et al.; Growth factors, hormones, and other regulatory molecules are traditionally required in tissue engineering studies to direct the differentiation of progenitor cells along specific lineages . We demonstrate that mechanical stimulation in vitro, without ligament-selective exogenous growth and differentiation factors, induces the differentiation of mesenchymal progenitor cells from the bone marrow into a ligament cell lineage in preference to alternative paths (i.e., bone or cartilage cell lineages) . A bioreactor was designed to permit the controlled application of ligament-like multidimensional mechanical strains (translational and rotational strain) to the undifferentiated cells embedded in a collagen gel . The application of mechanical stress over a period of 21 days up-regulated ligament fibroblast markers, including collagen types I and III and tenascin-C, fostered statistically significant cell alignment and density and resulted in the formation of oriented collagen fibers, all features characteristic of ligament cells . At the same time, no up-regulation of bone or cartilage-specific cell markers was observed.Appl Environ Microbiol, 2002 Jan, 68(1), 173 - 80Isolation and characterization of Novosphingobium sp . strain MT1, a dominant polychlorophenol-degrading strain in a groundwater bioremediation system; Tiirola MA et al.; A high-rate fluidized-bed bioreactor has been treating polychlorophenol-contaminated groundwater in southern Finland at 5 to 8 degrees C for over 6 years . We examined the microbial diversity of the bioreactor using three 16S ribosomal DNA (rDNA)-based methods: denaturing gradient gel electrophoresis, length heterogeneity-PCR analysis, and restriction fragment length polymorphism analysis . The molecular study revealed that the process was dependent on a stable bacterial community with low species diversity . The dominant organism, Novosphingobium sp . strain MT1, was isolated and characterized . Novosphingobium sp . strain MT1 degraded the main contaminants of the groundwater, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol, at 8 degrees C . The strain carried a homolog of the pcpB gene, coding for the pentachlorophenol-4-monooxygenase in Sphingobium chlorophenolicum . Spontaneous deletion of the pcpB gene homolog resulted in the loss of degradation ability . Phenotypic dimorphism (planktonic and sessile phenotypes), low growth rate (0.14 to 0.15 h(-1)), and low-copy-number 16S rDNA genes (single copy) were characteristic of strain MT1 and other MT1-like organisms isolated from the bioreactor.J Chromatogr A, 2001 Dec 14, 938(1-2), 67 - 77Recalcitrance of poly(vinylpyrrolidone): evidence through matrix-assisted laser desorption-ionization time-of-flight mass spectrometry; Trimpin S et al.; The aerobic biodegradability of an extensively used synthetic polymer was monitored the first time on a laboratory-scale fixed-bed bioreactor (FBBR) applying matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) . Polymeric poly(vinylpyrrolidone) (PVP) was spiked at concentrations of 10 mg l(-1) onto the FBBR run with river water and the biodegradation monitored after lyophilization of aliquots of the test liquor applying MALDI-TOF-MS . The latter proved to be a powerful tool for qualitative screening purposes of PVP in a molecular mass range <20 class="footer">g, e. The enhanced functional activity of hepatocytes seeded within the galactose modified scaffolds was likely caused by the formation of aggregated hepatocytes within the scaffolds . Biotechnol Bioeng, 2002 Jan 5, 77(1), 83 - 90A novel multi-coaxial hollow fiber bioreactor for adherent cell types . Part 1: hydrodynamic studies; Wolfe SP et al.; A novel multi-coaxial bioreactor for three-dimensional cultures of adherent cell types, such as liver, is described . It is composed of four tubes of increasing diameter placed one inside the other, creating four spatially isolated compartments . Liver acinar structure and physiological parameters are mimicked by sandwiching cells in the space between the two innermost semi-permeable tubes, or hollows fibers, and creating a radial flow of media from an outer compartment (ECC), through the cell mass compartment, and to an inner compartment (ICC) . The outermost compartment is created by gas-permeable tubing, and the housing is used to oxygenate the perfusion media to periportal levels in the ECC . Experiments were performed using distilled water to correlate the radial flow rate (Q(r)) with (1) the pressure drop (DeltaP) between the media compartments that sandwich the cell compartment and (2) the pressure in the cell compartment (P(c)) . These results were compared with the theoretical profile calculated based on the hydraulic permeability of the two innermost fibers . Phase-contrast velocity-encoded magnetic resonance imaging was used to visualize directly the axial velocities inside the bioreactor and confirm the assumptions of laminar flow and zero axial velocity at the boundaries of each compartment in the bioreactor . Axial flow rates were calculated from the magnetic resonance imaging results and were similar to the measured axial flow rates for the previously described experiments . Biotechnol Bioeng, 2001 Dec 20, 75(6), 691 - 701Application of redox mediators to accelerate the transformation of reactive azo dyes in anaerobic bioreactors; van der Zee FP et al.; Azo dyes are nonspecifically reduced under anaerobic conditions but the slow rates at which reactive azo dyes are converted presents a serious problem for the application of anaerobic technology as a first stage in the complete biodegradation of these compounds . As quinones have been found to catalyze reductive transfers by acting as redox mediators, the application of anthraquinone-2,6-disulfonic acid (AQDS) during continuous anaerobic treatment of the reactive azo dye, Reactive Red 2 (RR2), was evaluated . A mixture of volatile fatty acids was used as the electron-donating primary substrate . Batch experiments demonstrated that AQDS could increase the first-order rate constant of RR2 reductive cleavage by one order of magnitude . In the continuous experiment, treatment of RR2 containing synthetic wastewater in a lab-scale upflow anaerobic sludge blanket (UASB) reactor yielded low dye removal efficiencies (<30%)>90%) over a range of hydraulic retention times (HRT) (3-29 h) . Specifically, concentrations of total PAH, naphthalene, pyrene and total phenols in the feedstock and effluent of the SFFR were 123, 60, 51, 1.38 and 0.004, 0.001, 0.004, 0.1mg/l, respectively . The FBR was only marginally less effective than the SFFR for the same groundwater contaminants . Discharge to sewer was the most appropriate end use for the effluent . SFFRs are regarded as being simpler in design and operation, and a commercially available unit has been identified which would be suitable for treating small volumes (<10 class="footer" href="http://www.bionewsonline.com/o/r_paper.htm">l, a, e, i, k. The amounts of SRB and denitrabacteria were also higher in digester D5 than those in digesters D1, D2, D3 and D4 . Refuse decomposition could be accelerated by leachate recycle and inoculation in the view of microorganism development.Artif Organs, 2001 Sep, 25(9), 740 - 8Long-term expression of highly differentiated functions by isolated porcine hepatocytes perfused in a radial-flow bioreactor; Morsiani E et al.; To overcome the limitations of standard hollow-fiber module in ensuring efficient cell perfusion and long-term expression of highly differentiated hepatocyte functions, we developed a novel bioreactor in which a three-dimensional hepatocyte culture system was perfused in radial-flow geometry . Isolated porcine hepatocytes were cultured for 2 weeks in recirculating serum-free tissue culture medium, in which NH4Cl and lidocaine were repeatedly added, and ammonia removal, urea synthesis, monoethylglycinexylide (MEGX) production, albumin secretion, Po2, Pco2, O2 consumption, and pH were measured thereafter . During the whole duration of the study, ammonia removal was paralleled by urea production, while MEGX concentration was constantly increased . Our results indicated that hepatocytes remained differentiated and metabolically active throughout the duration of the study . The radial-flow bioreactor allowed physiological contact between recirculating fluid and cells by equalizing the concentration of the perfusing components, including O2, throughout the module, suggesting a potential use of this configuration for extracorporeal liver support.Artif Organs, 1994 Aug, 18(8), 611 - 7Culture of a differentiated liver cell line, Hep G2, in serum with application to a bioartificial liver: effect of supplementation of serum with amino acids; Melkonian AD et al.; Much effort has been directed toward the development of serum-free, hormonally defined culture conditions for the maintenance of differentiated functions in many cell types, including hepatocytes . However, in the development of a hepatocyte bioreactor for artificial liver support, many designs propose the maintenance of cells in plasma as opposed to defined culture medium . There is very little reported literature on the growth and function of cells cultured in plasma or serum; therefore, the effect of increasing serum concentrations was investigated using the human hepatoma, Hep G2, as a model cell line . It was found that Hep G2 can survive and grow in 100% serum if the serum is supplemented with L-glutamic acid, glycine, and L-cysteine.Anal Biochem, 1994 Aug 1, 220(2), 297 - 302On-line enzymatic amplification by substrate cycling in a dual bioreactor with rotation and amperometric detection; Raba J et al.; The amplification approach centered on the cycling of two reversibly interconvertible chemical species sequentially participating in two different enzyme-catalyzed reactions (enzymatic amplification by substrate cycling) has been implemented on-line into a continuous-flow/stopped-flow/continuous-flow operation . The implementation is illustrated with the determination of L-lactate in a dual enzyme reactor containing immobilized lactate oxidase (LOD, EC 1.1.3.x) to catalyze the oxidation of L-lactate by dissolved oxygen . The immobilized LOD was affixed to a rotating disk in the lower part of the flow-through cell . Immobilized lactate dehydrogenase (EC 1.1.1.27), affixed to the top part of the cell regenerates L-lactate with the mediation of beta-NADH as the hydrogen donor . The substrate cycling permits the generation of H2O2 beyond the stoichiometric limitation, and this is detected at a stationary Pt-ring electrode located at the bottom part of the cell . The stationary Pt-ring electrode is positioned concentrically to the rotating disk containing the immobilized LOD . The resulting amplified response permits, in a simple manner, achievement of detection limits as low as 0.3 fmol.liter-1 and allows the processing of 30 samples per hour.Biotechnol Appl Biochem, 1994 Aug, 20 ( Pt 1), 79 - 92Kinetic study of anaerobic digestion of fruit-processing wastewater in immobilized-cell bioreactors; Borja R et al.; The kinetics of the anaerobic digestion of a fruit-processing wastewater {chemical oxygen demand (COD) = 5.1 g/l} were investigated . Laboratory experiments were carried out in bioreactors containing supports of different chemical composition and features, namely bentonite and zeolite (aluminum silicates), sepiolite and saponite (magnesium silicates) and polyurethane foam, to which the microorganisms responsible for the process adhered . The influence of the support medium on the kinetics was compared with a control digester with suspended biomass . Assuming the overall anaerobic digestion process conforms to first-order kinetics, the specific rate constant, K0, was determined for each of the experimental reactors . The average values obtained were: 0.080 h-1 (bentonite); 0.103 h-1 (zeolite); 0.180 h-1 (sepiolite); 0.198 h-1 (saponite); 0.131 h-1 (polyurethane); and 0.037 h-1 (control) . The results indicate that the support used to immobilize the micro-organisms had a marked influence on the digestion process; the results were significant at the 95% confidence level . Methanogenic activity increased linearly with COD, with the saponite and sepiolite supports showing the highest values . The yield coefficient of methane was 270 ml of methane (under standard temperature and pressure conditions)/g of COD . The average elimination of COD was 89.5%.J Biotechnol, 1994 Jul 29, 36(1), 35 - 8A comparison of monoclonal antibody productivity in different hollow fiber bioreactors; Lowrey D et al.; Cell culture in hollow fiber bioreactors has been used as a method for large-scale production of monoclonal antibodies, viruses, cell-associated proteins and cancer antigens . We have examined an important variable in culturing cells in hollow fiber bioreactors: bioreactor composition . Eight different bioreactor designs which varied in nominal molecular weight cutoff, surface area, fiber material and ultra filtration rate were compared in large-scale hollow fiber cultures . A standard protocol utilizing the hybridoma 3C11 (ATCC HB 8511) or African green monkey kidney cells (Vero cells, ATCC CRL 1587) was designed so that the only variable examined was the hollow fiber bioreactor in use . The results suggest that surface area has little effect on antibody productivity, while fiber composition and ultrafiltration rate may play an important role . Vero cell growth was affected by both fiber composition and ultrafiltration rate.Int J Biochem, 1994 Jul, 26(7), 859 - 70Transgenic bioreactors; Janne J et al.; 1 . Although many human therapeutic proteins are currently produced in microbial fermentors using recombinant DNA techniques, it is obvious that microbial processing is not suitable for a large number of bioactive proteins owing to the inability of bacteria to carry out postsynthetic modification reactions required for full biological activity . 2 . This disadvantage does not apply to animal cell bioreactors that can generate biologically fully active entities, yet the use of large-scale animal cell cultures for production purposes is prohibitively expensive . 3 . With the advent of transgenic technology, the production of valuable human pharmaceuticals in large farm animals (pig, sheep, goat and dairy cattle) has become more and more attractive as a high-quantity, low-cost alternative . By employing targeted gene transfer, e.g . using mammary gland-specific regulatory sequences fused with the desired production genes, it is possible to govern the expression to occur exclusively in the mammary gland and hence the gene product is being ultimately secreted in the milk . 4 . While reviewing the remarkable progress in this field that has even led to commercial exploitations, we will outline in somewhat greater detail our strategy for the use of dairy cattle as a bioreactor for valuable proteins of pharmaceutical interest.Am J Physiol, 1994 Jul, 267(1 Pt 1), C195 - 20331P-MRS measurements of extracellular pH of tumors using 3-aminopropylphosphonate; Gillies RJ et al.; The extracellular pH (pHex) of tumors is generally acidic . However, it is only recently that noninvasive magnetic resonance spectroscopic (MRS) measurements have determined that the intracellular pH (pHin) of tumor cells in situ is neutral or slightly alkaline compared with that of normal tissues . Thus cells in tumors maintain larger pH gradients than do cells in nontumor tissues . To date, measurements of pHex in tumors have been made using microelectrodes, which preclude measurement of pHex and pHin within the same preparation . In addition, microelectrodes are invasive and have the potential to alter the measured pH values . The present communication describes simultaneous measurement of pHex and pHin in vitro in bioreactor culture and in vivo using 31P-MRS analyses of 3-aminopropylphosphonate (3-APP) and inorganic phosphate . In vitro results indicate that 3-APP is not toxic and that its resonant frequency is sensitive to pH and not significantly affected by temperature or ionic strength . Bioreactor experiments indicate that this compound is neither internalized nor metabolized by cells . Experiments in vivo indicate that 3-APP can be administered intraperitoneally and that RIF-1 tumors maintain a steady-state pHin of 7.25 and a pHex of 6.66 . These data have significance to basic tumor cell physiology and to the design of approaches to cancer chemotherapy and hyperthermic therapy, because both of these modalities exhibit pH sensitivity . It is also likely that these techniques will be applicable to localized MRS of other organ systems in vivo.Cell Transplant, 1994 Jul-Aug, 3(4), 263 - 71Expansion of activated lymphocytes obtained from renal cell carcinoma in an automated hollow fiber bioreactor; Hillman GG et al.; Immunotherapy using IL-2 alone or combined with activated lymphocytes has been promising for metastatic renal cell carcinoma . Cytotoxic lymphocytes can be isolated from tumors, expanded in vitro with IL-2, and adoptively transferred back into the tumor-bearing host . These cells can also be transduced with the genes coding for cytokines for local delivery to tumor sites . A major drawback in adoptive immunotherapy is the cumbersome and expensive culture technology associated with the growth of large numbers of cells required for their therapeutic effect . To reduce the cost, resources, and manpower, we have developed the methodology for lymphocyte activation and expansion in the automated hollow fiber bioreactor IMMUNO*STAR Cell Expander (ACT BIOMEDICAL, INC) . Tumor Infiltrating Lymphocytes (TIL) isolated from human renal cell carcinoma tumor specimens were inoculated at a number of 10(8) cells in a small bioreactor of 30 ml extracapillary space volume . We have determined the medium flow rates and culture conditions to obtain a significant and repeated expansion of TIL at weekly intervals . The lymphocytes cultured in the bioreactor demonstrated the same phenotype and cytotoxic activity as those expanded in parallel in tissue culture plates . Lymphocyte expansion in the hollow fiber bioreactor required lower volumes of medium, human serum, IL-2 and minimal labor . This technology may facilitate the use of adoptive immunotherapy for the treatment of refractory malignancies.Braz J Med Biol Res, 1994 Jul, 27(7), 1575 - 87Studies on the efficiency of measles virus antigen production using VERO cell culture in a microcarrier system; Mendonca RZ et al.; 1 . A large amount of antigen is required to conduct seroepidemiologic surveys of measles . Thus, a process to obtain measles virus antigen using a bioreactor was standardized . 2 . The virus was grown in a 3.7-1 culture of VERO cells using a Celligen cell culture system containing 2 mg/ml of microcarriers (cytodex I) at 37 degrees C and 60 rpm . The cultures infected with 0.5 m.o.i . of measles virus were harvested after the appearance of the cytopathic effect . The virus suspension was clarified and concentrated by ultracentrifugation . Intracellular and extracellular virus titers were determined by hemagglutination (HA) and by induction of a cytopathic effect in cell culture (TCID50) . 3 . Intracellular virus presented 5-7 x 10(6) TCID50/0.1 ml, HA activity per 50 microliters equal to 32, with a total HA activity of 4,480 HA units (HAU) and specific activity of 116 HAU/mg . In the concentrated supernatants, the HA titer of extracellular virus was 64, with a total HA activity of 1,024 HAU and a specific activity of 1,600 HAU/mg . 4 . The antigen obtained was suitable for the detection of antibodies against measles virus in assays such as ELISA and DOT-ELISA (using 1 micrograms/well to ELISA and 2 micrograms/DOT) . 5 . The microcarrier system produced antigen sufficient for 26 ELISAs/ml compared to 5.7 ELISAs/ml obtained for the static culture system.Biotechnol Prog, 1994 Jul-Aug, 10(4), 410 - 20Cultivation of recombinant baby hamster kidney cells in a fluidized bed bioreactor system with porous borosilicate glass; Kratje RB et al.; Dense cell cultivation of the recombinant cell line BHK 21 pSVIL2 was performed in a fluidized bed bioreactor system containing porous borosilicate glass carriers . Experiments were carried out with different medium formulations for a period of 48 days . Due to an effective immobilization of the cells in the reactor, continuous operation was easy to perform . Maximal cell densities and product yields could be maintained, even when protein-free medium was perfused exceeding 2 reactor volumes per day . Final cell densities of magnitude 7.1 x 10(7) mL-1 intrasphere volume were reached, while the interleukin-2 production rate was 0.70 mg day-1 . The cell specific productivity reached a value of 1.3 x 10(-10) mg day-1 . The first results were presented with a cell line that grows under glutamine-free medium conditions . The use of a glutamine-free medium for the cultivation of the cells resulted in a drastic decrease in cell metabolism . Furthermore, the amino acids lysine and histidine were produced and secreted into the culture supernatant, although these metabolites normally are considered to be essential for animal cells grown in vitro . However, no lethal effect on the cells has been detected, and the total number of cells in the reactor remained constant . The metabolism of threonine has been detected to be directly dependent on the presence of glutamine . Cells grown in glutamine-free culture medium produced glycine yields 6 times higher than those grown in glutamine-containing medium . A bead-to-bead transfer of the cells has also been detected when the cells immobilized within the intrasphere volume of the borosilicate carriers reached the stationary phase.Appl Microbiol Biotechnol, 1994 Jul, 41(5), 578 - 83Overproduction of mannitol dehydrogenase in Rhodobacter sphaeroides; Schneider KH et al.; Mannitol dehydrogenase (MDH) from Rhodobacter sphaeroides Si4 was overproduced by constructing a strain that overexpresses the MDH gene and by producing high cell concentrations via fed-batch cultivation in a bioreactor . With the gene of mannitol dehydrogenase (mtlK) cloned into the expression vector pKK223-3 expression of MDH in Escherichia coli was obtained, but the specific enzyme activity was lower than in R . sphaeroides Si4 . In order to overexpress mtlK in R . sphaeroides, plasmid pAK82 was constructed by cloning a DNA fragment carrying mtlK into the broad-host-range expression vector pRK415 . When pAK82 was introduced into R . sphaeroides Si4 the specific mannitol dehydrogenase activity in the strain obtained was 0.48 unit (U)mg-1,3.4-fold higher than in the wild type . In this way the enzyme yield from cultivation in a bioreactor could be improved from 110 Ul-1 to 350 Ul-1 . A further increase in productivity was obtained by fed-batch cultivation of R . sphaeroides Si4 {pAK82} . Using this cultivation method an optical density of 27.6 was reached in the bioreactor, corresponding to a dry mass of 16.6 g l-1 . Since MDH formation correlated with biomass production, the MDH yield could be raised to 918 Ul-1, an 8.3-fold increase in comparison to batch cultivation of the wild-type strain.J Chem Technol Biotechnol, 1994 Jul, 60(3), 327 - 34Kinetics of anaerobic digestion of soft drink wastewater in immobilized cell bioreactors; Borja R et al.; A kinetic study of the anaerobic digestion of soft drink wastewater was undertaken, using bioreactors containing various suspended supports (bentonite, zeolite, sepiolite, saponite and polyurethane foam), on to which the microorganisms effecting the purification were immobilized . Assuming the overall anaerobic digestion process conforms to first-order kinetics, the specific rate constants, K0, derived from the reactors with saponite and sepiolite (magnesium silicates) were approximately twice those from bentonite and zeolite (aluminium silicates) and almost five times higher than in the control reactor (without support); the polyurethane support showed an intermediate behaviour . The methanogenic activity increased linearly with COD load, with saponite and sepiolite supports showing the highest values . The average yield coefficient of methane was 325 cm3 CH4 STP g-1 COD and the percentage elimination of COD was 77.8%; these values were not significantly altered by the type of support used.J Biotechnol, 1994 Jun 15, 35(1), 1 - 7Large-scale cultivation of Catharanthus roseus cells: production of ajmalicine in a 20-l airlift bioreactor; Fulzele DP et al.; Bioreactor systems have been developed for the production of ajmalicine, an alkaloid used in the treatment of hypertension . Cell cultures of Catharanthus roseus produced higher levels of ajmalicine (323 micrograms g-1 dry weight) in a production medium enriched with tryptophan . The cell cultures were grown in medium prepared in tap water and market sugar with a view to minimise the costs of production . Large-scale cultivation of cell suspension was performed in a 20-l airlift bioreactor under controlled conditions . An ajmalicine production of 315 micrograms g-1 dry weight was achieved in the bioreactor after 14 d of cultivation.Chin Med Sci J, 1994 Jun, 9(2), 71 - 4High density cultivation of genetically-engineered CHO cell lines with microcarrier culture systems; Xiao C et al.; Genetically-engineered CHO cell lines, r beta-13 and CLF-8B2, were cultivated with the MC-1 microcarrier culture system . The cell density could be enhanced by increasing the concentration of microcarrier . At a microcarrier concentration of 10 mg/ml, the cell density could reach 4 to 5 x 10(6) cells/ml . It was shown that these cell lines would spontaneously release from the microcarrier to attach to and proliferate on fresh microcarriers . We were thus able to scale up cultivation using a simple method, i.e . by adding fresh microcarriers and medium directly into the culture system to about 2, 4 or 8 times the original volume . Using a perfusion culture system, we have successfully cultivated CLF-8B2 cells in a 2 L bioreactor for several weeks at medium perfusion rates of 0.5 to 3 working volumes . Prourokinase was stably secreted.Enzyme Microb Technol, 1994 Jun, 16(6), 506 - 12Optimization of the growth conditions of Sf21 insect cells for high-density perfusion culture in stirred-tank bioreactors; Deutschmann SM et al.; Spodoptera frugiperda insect cells (IPLB-Sf21-AE) (Sf21), infected with baculovirus expression vectors during their exponential growth phase, are commonly used to produce a variety of heterologous recombinant proteins . In the present study the culture conditions of these insect cells were studied to establish high-density suspension cultures with prolonged exponential growth phases . The Sf21 cells were grown in 125-ml spinner flasks using five different culture media supplemented with 5% fetal calf serum and four protein-free or low-protein culture media . The best results were achieved in EX-CELL 401 (protein-free media) and in IPL-41 modified with 2.5 g l-1 tryptose phosphate broth (serum-supplemented media), respectively . The latter was used for further batch and continuous cultivation of Sf21 cells in a perfused 1.4-l stirred-tank bioreactor with special attention to the oxygen requirement of these cells. g, d, j, j, g. Optimal growth was found at an oxygen concentration of 70% air saturation, resulting in a prolonged exponential growth phase that could be maintained for more than 16 days . A maximum cell density of 5.5 x 10(7) viable cells ml-1 was achieved.Hum Cell, 1994 Jun, 7(2), 95 - 100{A new liver support system composed of functional human cells and a radial-flow bioreactor}; Kawada M et al.; An artificial liver will be useful for the treatment of acute hepatic failure and a bridge of liver transplantation . The current reports suggest that the hybrid type of artificial liver composed of functional human liver cells and a bioreactor is practical for clinical use . In the present study, we succeeded high density culture on a large-scale of human functional hepatoma (JHH-7) using a newly developed radial flow packed-bed bioreactor . Since the shear stress of this bioreactor is lower than the other type, high density culture without cell damage is possible . JHH-7 cells produced large amounts of human albumin and other liver specific proteins, and then have the function of ammonia metabolism in the system . This study suggests that a radial flow bioreactor will be developed as a new type of artificial liver.J Biotechnol, 1994 May 31, 34(3), 247 - 57Re-use of spent cell culture medium in pilot scale and rapid preparative purification with membrane chromatography; Riese U et al.; Based on experiments in bench scale, a recycling of spent cell culture medium was performed in a 100-1 pilot scale bioreactor . The cell cultivation has been done as a repeated batch procedure after the initial batch in the following four repeated batches spent medium from the previous batch was partially re-used . After microfiltration and ultrafiltration a part of the filtrate was mixed with a concentrate of amino acids and glucose, sterile filtered and subsequently filled back into the bioreactor . Up to 65% of the harvested cell- and product-free spent medium was re-used in each repeated batch . This procedure results in a saving of pure and waste water volume and saving of supplemented proteins as transferrin, insulin and lipoproteins and, therefore, also in a reduction of the production costs . A strongly acidic membrane ion exchanger was evaluated for the ability to purify the monoclonal antibodies from the pilot scale cultivation . Within minutes, gram quantities of product could be purified in a high flux system, especially developed for this purpose, achieving purities of 80% . The capacity of the acidic membrane ion exchanger was found in former investigations to be 1 mg cm-2 with recoveries up to 96% . Final purification was carried out by gel column filtration.