ROLE OF BIOREACTOR IN BIOPROCESS ENGG.-02

Biotechnol Bioeng . 2004 Dec 23; {Epub ahead of print}Protein production and induction of the unfolded protein response in Trichoderma reesei strain Rut-C30 and its transformant expressing endoglucanase I with a hydrophobic tag; Collen A et al.; The effect of induction of protein production was studied in bioreactor cultures of T . reesei strain Rut-C30 and its transformant expressing endoglucanase I core domain (EGI, Cel7B) fused with a hydrophobic peptide tag . The tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation . The fungi were first grown on glucose-containing minimal medium after which rich medium with lactose as a carbon source was added to induce cellulase production . Production of extracellular protein and cellulase activity and the transcript levels of the major cellulase genes were analyzed during the cultivations . Induction of the cellulase genes followed a similar temporal pattern in both strains . The first phase of induction took place after addition of lactose as soon as glucose was depleted, and the second phase after lactose was consumed . Western analysis showed that a decreased amount of fusion protein was produced in the culture medium compared with the endogenous EGI, although the strain harbors several copies of the recombinant gene under the strong cbh1 promoter . The fusion protein appeared to accumulate within the cells, indicating impaired secretion of the protein . The mRNA levels of the UPR (unfolded protein response) target genes, bip1 and pdi1, and the level of the active form of hac1 transcript encoding the UPR transcription factor increased concurrently with induction of the cellulase genes in both strains, indicating increased requirement of the folding machinery under these conditions . However, only a minor increase in bip1 and pdi1 transcript level was observed in the transformant compared with the parental strain . (c) 2004 Wiley Periodicals, Inc.J Agric Food Chem, 2004 Dec 29, 52(26), 7842 - 5A novel angiotensin I converting enzyme inhibitory peptide from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate; Je JY et al.; Alaska pollack frame protein, which is normally discarded as an industrial byproduct in the processing of fish in plants, was hydrolyzed with pepsin . This was fractionated into five major types of Alaska pollack frame protein hydrolysates (APH-I, 10-30 kDa; APH-II, 5-10 kDa; APH-III, 3-5 kDa; APH-IV, 1-3 kDa; and APH-V, below 1 kDa) using an ultrafiltration membrane bioreactor system . Angiotensin I converting enzyme (ACE) inhibitory activities of the fractionated hydrolysates were investigated, and the fraction that exhibited the highest ACE inhibitory activity was further purified using consecutive chromatographic methods on SP-Sephadex C-25 column, Sephadex G-25 column, and high-performance liquid chromatography (HPLC) on an octadecylsilane column . Finally, we purified a novel ACE inhibitory peptide with an IC50 value of 14.7 microM, and the sequence of the peptide was Phe-Gly-Ala-Ser-Thr-Arg-Gly-Ala . In addition, the ACE inhibition pattern of the peptide was found to be noncompetitive.Biotechnol Bioeng . 2004 Dec 17; {Epub ahead of print}Culture of Escherichia coli under dissolved oxygen gradients simulated in a two-compartment scale-down system: Metabolic response and production of recombinant protein; Sandoval-Basurto EA et al.; A significant problem of large-scale cultures, but scarcely studied for recombinant E . coli, is the presence of gradients in dissolved oxygen tension (DOT) . In this study, the effect of DOT gradients on the metabolic response of E . coli and production of recombinant pre-proinsulin, accumulated as inclusion bodies, was determined . DOT gradients were simulated in a two-compartment scale-down system consisting of two interconnected stirred-tank bioreactors, one maintained at anoxic conditions and the other at a DOT of at least 6% . Cells were continuously circulated between both vessels to simulate circulation times (t(c)) of 20, 50, 90, and 180 sec . A complete kinetic and stoichiometric characterization was performed in the scale-down system as well as in control cultures maintained at constant DOT in the range of 0-20% . The performance of E . coli cultured under oscillating DOT was significantly affected, even at a t(c) of 20 sec corresponding to transient exposures of only 13.3 sec to anaerobic conditions . Specific growth rate decreased linearly with t(c) to a maximum reduction of 30% at the highest t(c) tested . The negative effect of DOT gradients was even more pronounced for the overall biomass yield on glucose and the maximum concentration and yield of pre-proinsulin . In these cases, the losses were 9%, 27%, and 20%, respectively, at t(c) of 20 sec and 65%, 94%, and 87%, respectively, at t(c) of 180 sec . Acetic, lactic, formic, and succinic acids accumulated during oscillatory DOT cultures, indicating that deviation of carbon flow to anaerobic metabolism was responsible for the observed losses . The results of this study indicate that even very short exposures to anaerobic conditions, typical of large-scale operations, can substantially reduce recombinant protein productivity . The information presented here is useful for establishing improved rational scale-up strategies and understanding the behavior of recombinant E . coli exposed to DOT gradients . (c) 2004 Wiley Periodicals, Inc.J Ind Microbiol Biotechnol . 2004 Dec 17; {Epub ahead of print}Cometabolic reduction of bromate by a mixed culture of microorganisms using hydrogen gas in a gas-lift reactor; Ginkel CG et al.; The discharge of bromate, a suspected carcinogen, will be restricted in the near future . To assess the possibility of biotechnological treatment of bromate-containing wastewaters, the removal of bromate by chlorate-reducing microorganisms was studied . The removal of bromate and chlorate was studied in laboratory gas-lift bioreactors supplied with hydrogen gas as electron donor in the absence of molecular oxygen . In these reactors, bromate was reduced cometabolically by chlorate-respiring microorganisms . To allow the cometabolic reduction of bromate, a chlorate:bromate molar ratio of at least 3:1 was required . The cometabolic conversion permitted almost complete reduction of bromate into bromide at hydraulic retention times of at least 6 h . Optimal bromate reduction activity was observed at approximately 35 degrees C . The pH optimum was between 7 and 8 . Bromate reduction in excess of 80% and a maximum bromate reduction rate of 2.3 g l(-1) day(-1) in a pilot-scale gas-lift bioreactor demonstrates that the process is sustainable.Biotechnol Lett, 2004 Nov, 26(21), 1619 - 22Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate; Kim YS et al.; Adventitious roots of ginseng were treated with methyl jasmonate (MJ) up to 150mu and cultured for 40days . Up to 100mu MJ inhibited the root growth but increase ginsenoside accumulation . In a two-stage bioreactor culture, total ginsenosides, after elicitation with 100 mu MJ peaked after 10days at 48mgg(-1) dry wt and then dropped sharply . Of the two groups of ginsenosides (Rb and Rg), higher amounts of Rb accumulated in the adventitious roots.Altern Lab Anim, 2004 Mar, 32(1), 25 - 35Evaluation of a flat membrane hepatocyte bioreactor for pharmacotoxicological applications: evidence that inhibition of spontaneously produced nitric oxide improves cell functionality; Canova N et al.; A laboratory-scale bioreactor was re-evaluated, with the aim of improving its use for the perfused culture of rat hepatocytes . In contrast to conventional culture systems, the flat membrane bioreactor (FMB) showed good functionality and biochemical competence during 2-3 days . Hepatocytes cultured in the FMB, specifically in a "sandwich" configuration, were functionally stable, as shown by a high rate of urea biosynthesis after challenge with NH4Cl, a low alanine-aminotransferase leakage and suppressed spontaneous nitric oxide (NO) production . Moreover, the time-course of the disappearance of cyclosporin A (CsA) from the perfusate demonstrated the high biotransformation capacity of cells in the FMB . The effect of CsA on the modulation of urea and spontaneous NO production demonstrated flexibility, in that minor changes could be observed at diverse time intervals and in a non-destructive way . The monitoring of nitrite levels during various steps of isolation and culture suggested that spontaneously produced NO has a negative impact on hepatocyte metabolic and functional integrity . In spite of the sophisticated techniques that are being used for the preparation of bioreactors, with hepatocytes surviving for longer periods, our data have shed light on some factors that could be important for the successful use of similar models for pharmacotoxicological and other biomedical applications.J Neurosci Res, 2005 Jan 1-15, 79(1-2), 26 - 32Culturing primary brain astrocytes under a fully controlled environment in a novel bioreactor; Sa Santos S et al.; We report the first approach for growth and maintenance of primary astrocytes on a fully controlled environment . For this purpose, cells were immobilized in Cytodex microcarriers and grown in a stirred tank bioreactor . The distribution of astrocytes at the microcarrier surface was visualized using confocal microscopy and glial fibrillary acidic protein (GFAP) labeling, a specific glial probe . Crucial bioreaction parameters such as agitation rate, microcarrier type, and concentration, as well as cell inoculum concentration were assessed . Cytodex 3 proved the best microcarrier for astrocyte growth, with the highest cell densities obtained for 6 g/l of Cytodex 3 using an inoculum of approx . 0.15 x 10(6) cells/ml in vessels operated at 60 rpm, using a refeed operational mode consisting of complete medium replacement every 5 days . Using such optimized conditions, cells were maintained in steady-state for approximately 24 days, allowing online monitoring and control of environmental variables such as temperature, pH, and O(2) . To test further the advantages of this fully controlled system, astrocytes were also subjected to hypoxic stress for 5 hr; the cell number was not affected by hypoxia but the glycolytic flux was enhanced during the stress imposed . The culture system described is a novel tool to study brain cell metabolism, allowing sampling over time and the monitoring of cellular behavior through stressful conditions and during recovery . (c) 2004 Wiley-Liss, Inc.Hum Antibodies, 2004, 13(3), 69 - 79Production of a human monoclonal IgM directed against human cardiac myosin in a hollow-fiber bioreactor for Membrane Anion Exchange Chromatography one-step purification; Jacobin MJ et al.; Purification of human IgM monoclonal antibodies (MAbs) has proved to be difficult . Since IgM Mabs tend to bind strongly to a variety of resin support surfaces, the number of chromatographic steps used in the purification of these biomolecules should be minimized . Here we describe procedures developed for the optimal production and purification of the human monoclonal IgM B7, which specifically binds to the myosin heavy chain of human ventricular myocardium . This property makes this antibody potentially useful for the diagnosis of myocardial necrosis . Several chromatographic techniques were evaluated (size exclusion, ion exchange, affinity chromatography) . The best results were obtained with anion exchange membrane chromatography using Sartobind Q15 (98% purity, 30% recovery) . IgM production was improved by the hollow fiber technology which permitted the use of serum-reduced medium and an increase in antibody concentration to an average production of 300-400 microg/ml, compared to 20 microg/ml in flask culture . Several flow-rates were also evaluated, the optimal being 20 ml/minute for 30% of recovery . Importantly, the purified IgM molecule was able to bind to human myosin in ELISA and Western-blotting, thus allowing the IgM to be kept intact for further radiolabeling.Methods Mol Biol, 2005, 295, 55 - 70Growing hybridomas; Entrican G et al.; Hybridomas can be grown in a number of ways to produce stocks of monoclonal antibodies . Smaller volumes may be produced in static flask culture, but if larger amounts are required, a bioreactor may be used.Protein Expr Purif, 2005 Jan, 39(1), 61 - 70Optimisation of insect cell growth in deep-well blocks: development of a high-throughput insect cell expression screen; Bahia D et al.; This report describes a method to culture insects cells in 24 deep-well blocks for the routine small-scale optimisation of baculovirus-mediated protein expression experiments . Miniaturisation of this process provides the necessary reduction in terms of resource allocation, reagents, and labour to allow extensive and rapid optimisation of expression conditions, with the concomitant reduction in lead-time before commencement of large-scale bioreactor experiments . This therefore greatly simplifies the optimisation process and allows the use of liquid handling robotics in much of the initial optimisation stages of the process, thereby greatly increasing the throughput of the laboratory . We present several examples of the use of deep-well block expression studies in the optimisation of therapeutically relevant protein targets . We also discuss how the enhanced throughput offered by this approach can be adapted to robotic handling systems and the implications this has on the capacity to conduct multi-parallel protein expression studies.Toxicol Sci . 2004 Dec 8; {Epub ahead of print}In Vitro Zonation and Toxicity in a Perfused Hepatocyte Co-Culture; Allen JW et al.; In vitro models that more closely represent the intricate architecture and functional diversity of the liver would be beneficial to toxicology . We have established a bioreactor culture system that recapitulates features of liver zonation in vitro, allowing investigation of compartmentalized drug metabolism and toxicity . In vitro zonation is induced by exposing hepatocyte cultures to oxygen and nutrient gradients in a parallel plate bioreactor . Gradients were modeled and experimentally validated over co-cultures of hepatocytes and fibroblasts that exhibit a stable hepatocyte phenotype in vitro . Co-cultures exposed to physiologic oxygen gradients exhibited zonal induction of CYP2B and CYP3A protein that mimics that found in vivo . Furthermore, exposure to a prototypic zonal hepatotoxin, APAP, resulted in maximal toxicity at the low-oxygen outlet region similar to centrilobular necrotic patterns observed in vivo . In conclusion, we have established a perfused hepatocyte co-culture system for molecular and applied investigation into zonation-dependent phenomena involving drug metabolism and toxicity.Tissue Eng, 2004 Sep-Oct, 10(9-10), 1436 - 45Tribology approach to the engineering and study of articular cartilage; Wimmer MA et al.; This study has been based on the assumption that articular motion is an important aspect of mechanotransduction in synovial joints . For this reason a new bioreactor concept, able to reproduce joint kinematics more closely, has been designed . The prototype consists of a rotating scaffold and/or cartilage pin, which is pressed onto an orthogonally rotating ball . By oscillating pin and ball in phase difference, elliptical displacement trajectories are generated that are similar to the motion paths occurring in vivo . Simultaneously, dynamic compression may be applied with a linear actuator, while two-step-motors generate the rotation of pin and ball . The whole apparatus is placed in an incubator . The control station is located outside . Preliminary investigations at the gene expression level demonstrated promising results . Compared with free-swelling control and/or simply compression-loaded samples, chondrocyte-seeded scaffolds as well as nasal cartilage explants exposed to interface motion both showed elevated levels of cartilage oligomeric matrix protein mRNA . The final design of the bioreactor will include four individual stations in line, which will facilitate the investigation of motion-initiated effects at the contacting surfaces in more detail.Biomaterials, 2005 May, 26(15), 2509 - 16An organic-inorganic hybrid scaffold for the culture of HepG2 cells in a bioreactor; Kataoka K et al.; Much interest has recently been shown in the potential utility of bioartificial liver (BAL) as a bridge support for patients and as a module for experimental purposes . A radial-flow bioreactor (RFB), one of the perfused bed/scaffold-type bioreactors, enables a highly functional three-dimensional culture as BAL . The functional capacity of bioreactors depends not only on their mechanistic structures but also on scaffolds packed in them . In the present study, we examined the possible utility of a new porous organic-inorganic-hybrid scaffold in an RFB . The scaffold was made from tetraethoxysilane (TEOS) and polydimethylsiloxane (PDMS) by a sol-gel method using sieved sucrose particles as a porogen . In the porous TEOS-PDMS hybrid scaffold, human hepatocellular carcinoma cells (HepG2) proliferated actively and formed cell clusters more efficiently than they did in a polyvinyl-alcohol scaffold . When cultivated in PDMS-TEOS, HepG2 cells secreted a approximately three-fold greater amount of albumin than that secreted in a monolayer culture . For potential application of BAL to pharmacological studies and future clinical use, it is essential to develop a method to propagate liver cells that maintain highly specific functions . The present results indicate that PDMS-TEOS may be a promising scaffold for developing such functional culture methods.Biotechnol Bioeng, 2005 Jan 20, 89(2), 138 - 147Development of a small-scale bioreactor: Application to in vivo NMR measurement; Gmati D et al.; A perfused bioreactor allowing in vivo NMR measurement was developed and validated for Eschscholtzia californica cells . The bioreactor was made of a 10-mm NMR tube . NMR measurement of the signal-to-noise ratio was optimized using a sedimented compact bed of cells that were retained in the bioreactor by a supporting filter . Liquid medium flow through the cell bed was characterized from a mass balance on oxygen and a dispersive hydrodynamic model . Cell bed oxygen demand for 4 h perfusion required a minimal medium flow rate of 0.8 mL/min . Residence time distribution assays at 0.8-2.6 mL/min suggest that the cells are subjected to a uniform nutrient environment along the cell bed . Cell integrity was maintained for all culture conditions since the release of intracellular esterases was not significant even after 4 h of perfusion . In vivo NMR was performed for (31)P NMR and the spectrum can be recorded after only 10 min of spectral accumulation (500 scans) with peaks identified as G-6P, F-6P, cytoplasmic Pi, vacuolar Pi, ATP(gamma) and ADP(beta), ATP(alpha) and ADP(alpha), NADP and NDPG, NDPG and ATP(beta) . Cell viability was shown to be maintained as (31)P chemical shifts were constant with time for all the identified nuclei, thus suggesting constant intracellular pH . (c) 2004 Wiley Periodicals, Inc.Biotechnol Bioeng, 2005 Jan 20, 89(2), 157 - 63Low-temperature pausing of cultivated mammalian cells; Hunt L et al.; There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37 degrees C and freezing in small batches . We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37 degrees C in a variety of cultivation vessels . High cell viability and exponential growth were observed after pausing a recombinant Chinese hamster ovary cell line (CHO-Clone 161) in a temperature range of 6-24 degrees C in microcentrifuge tubes for up to 3 weeks . After pausing in T-flasks at 4 degrees C for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37 degrees C . Adherent cultures of CHO-DG44 cells paused for 2 days at 4 degrees C in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4 degrees C or 24 degrees C in spinner flasks were efficiently transfected by the calcium phosphate-DNA coprecipitation method, yielding reporter protein levels comparable to those from nonpaused cultures . Finally, cultures of a recombinant CHO cell line (CHO-YIgG3) paused for 3 days at 4 degrees C, 12 degrees C, or 24 degrees C in bioreactors achieved the same cell mass and recombinant protein productivity levels as nonpaused cultures . The success of this approach to cell storage with rodent and human cell lines points to a general biological phenomenon which may have a wide range of applications for cultivated mammalian cells . (c) 2004 Wiley Periodicals, Inc.J Hepatol, 2004 Dec, 41(6), 950 - 6Development of a scaled up liver device incorporating cryo-preserved pig liver micro-organs; Gershonowitz A et al.; BACKGROUND/AIMS: Currently there is no effective therapy for most patients with fulminant or end stage liver disease . METHODS: Pig liver micro-organs (LMOs), which preserve liver micro-architecture and ensure a maximal 150-200mum distance from a source of nutrients and gases have been prepared and a method to cryo-preserve them has been developed . A new scaled-up extra-corporeal liver device termed aLIVE-H in which LMOs are exposed to liver-like hemodynamic conditions has also been developed . The purpose of this work is to test the safety and function of cryo-preserved LMOs and how the hemodynamic properties of the scaled up aLIVE device affect their function . RESULTS: Pig LMOs in aLIVE-H, transcribe albumin and Factor V at similar levels, irrespective of their position within the bioreactor, indicating that the hemodynamic features of the aLIVE-H device allow for homogeneous plasma distribution and proper function at different locations . Cryo-preserved LMOs transcribe albumin and Factor V at levels comparable to those transcribed by a normal pig liver . Connecting the aLIVE-H bioreactor to normal pigs did not affect key blood components and biochemical parameters . CONCLUSIONS: An extra-corporeal liver device aLIVE-H which imitates the hemodynamic and functional properties of the normal liver and incorporates cryo-preserved LMOs has been developed and characterized . aLIVE-H was found to perform key synthetic liver functions.Chemosphere, 2005 Jan, 58(3), 299 - 310Carbon/electron source dependence of polychlorinated biphenyl dechlorination pathways for anaerobic granules; Nollet H et al.; The effect of acclimating anaerobic granules from commercial bioreactors with different carbon/electron sources on their ability to reductively dechlorinate a tri-(2,3,4-CB) and heptachlorobiphenyl (2,2',3,3',4,5,6-CB) was studied . The anaerobic granules were first grown in upflow anaerobic sludge blanket (UASB) reactors fed with two different mixtures of carbon/electron sources, i.e., propionate/butyrate/methanol and formate/methanol . Differences in dechlorination patterns for 2,2',3,3',4,5,6-CB were observed in batch experiments inoculated with granules from these two sets of UASB reactors . Variation of the carbon/electron source, during the dechlorination process, had no effect on the dechlorination pathway, but the extents and rates of dechlorination were highest for ethanol and formate and lowest for pyruvate fed batches . Pre-acclimation of different anaerobic sludges to polychlorinated biphenyls (PCBs) shortened the lag period, but did not influence the PCB dechlorination pathway . This is the first time that similar acclimation conditions for several anaerobic microbial communities prior to inoculation were reported to yield similar substrate specificities for the reductive dechlorination of specific PCB congeners . This research demonstrates a successful strategy for the development of biocatalysts to serve as the inoculum of partially decontaminated sites in order to provide microorganisms with specificities complementary to those of naturally occurring dechlorinators.Water Sci Technol, 2004, 50(9), 17 - 23Use of microwave pretreatment for enhanced anaerobiosis of secondary sludge; Park B et al.; This work elucidates the effects of pretreatment of secondary sludge by microwave irradiation on anaerobic digestion . The soluble chemical oxygen demand (COD) concentration increased up to 22% as microwave irradiation time increased, which indicated the sludge particles disintegrated . Three identical automated bioreactors with working volume of 5 l were used as anaerobic digesters at mesophilic temperature (35 degrees C) . The reactors were separately fed with sludge with microwave pretreated- and control- sludge at different hydraulic retention times (HRT) . The volatile solid (VS) reduction in the control operation was approximately 23.2 +/- 1.3%, while it was 25.7 +/- 0.8% for the reactors with the pretreated sludge . The average biogas production rate with the pretreated sludge at 8, 10, 12, and 15 days HRTs was 240 +/- 11, 183 +/- 9, 147 +/- 8, and 117 +/- 7 ml/l/d respectively, while those with the control sludge were 134 +/- 12 and 94 +/- 7 ml/l/d at 10 and 15 days HRTs . Maximum rates of COD removal and methane production with the pretreated sludge were 64% and 79% higher than those of the control system, respectively.Appl Microbiol Biotechnol . 2004 Dec 2; {Epub ahead of print}Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of Panax ginseng in 5-l balloon type bubble bioreactors; Thanh NT et al.; The effects of methyl jasmonate (MJ) elicitation on the cell growth and accumulation of ginsenoside in 5-l bioreactor suspension cultures of Panax ginseng were investigated . Ginsenoside accumulation was enhanced by elicitation by MJ (in the range 50-400 muM); however, fresh weight, dry weight and growth ratio of the cells was strongly inhibited by increasing MJ concentration . The highest ginsenoside yield was obtained at 200 muM MJ . In the second experiment, 200 muM MJ was added on day 15 during the cultivation . The ginsenoside, Rb group, and Rg group ginsenoside content increased 2.9, 3.7, and 1.6 times, respectively, after 8 days of MJ treatment . Rb group gisnsenosides accumulated more than Rg group ginsenosides . Among Rb group ginsenosides, Rb1 content increased significantly by four times but the contents of Rb2, Rc and Rd increased only slightly . Among Rg group ginsenosides, Rg1 and Re showed 2.3-fold and 3.0-fold increments, respectively, whereas there was only a slight increment in Rf group ginsenosides . These results suggest that MJ elicitation is beneficial for ginsenoside production using 5-l bioreactor cell suspension cultures.Biomaterials, 2005 May, 26(14), 2001 - 12Low-density cultures of bovine chondrocytes: effects of scaffold material and culture system; Hu JC et al.; Chondrocytes were seeded on either agarose or polyglycolic acid (PGA) unwoven meshes at 10 million cells/ml of scaffold volume to evaluate the effect that these two biomaterials have on the low-density culture of chondrocytes in a rotating-wall bioreactor . For both static and bioreactor culture, agarose constructs contained more glycosaminoglycan than their PGA counterparts . However, the PGA constructs contained more collagen for both culture conditions when compared to agarose . For the low seeding density of this study, PGA constructs cultured in the bioreactor did not outperform static cultures when comparing collagen content after 8 weeks . The mechanical properties of the PGA constructs also did not improve with culture time . Similar results were observed with the agarose culture, though both static- and bioreactor-culture agarose constructs exhibited increases in aggregate modulus at the end of the culture period . As in PGA culture, chondrocytes cultured in agarose may require a higher density to reap the benefits of the bioreactor environment.Biomaterials, 2005 May, 26(14), 1857 - 75The roles of tissue engineering and vascularisation in the development of micro-vascular networks: a review; Kannan RY et al.; The construction of tissue-engineered devices for medical applications is now possible in vitro using cell culture and bioreactors . Although methods of incorporating them back into the host are available, current constructs depend purely on diffusion which limits their potential . The absence of a vascular network capable of distributing oxygen and other nutrients within the tissue-engineered device is a major limiting factor in creating vascularised artificial tissues . Though bio-hybrid prostheses such as vascular bypass grafts and skin substitutes have already been developed and are being used clinically, the absence of a capillary bed linking the two systems remains the missing link . In this review, the different approaches currently being or that have been applied to vascularise tissues are identified and discussed.Biotechnol Prog, 2004 Nov-Dec, 20(6), 1888 - 92Temperature effects on product-quality-related enzymes in batch CHO cell cultures producing recombinant tPA; Clark KJ et al.; Culture conditions that affect product quality are important to the successful operation and optimization of bioreactors . Previous studies have demonstrated that enzymes, such as proteases and sialidases, accumulate in batch bioreactors . These enzymes are known to be detrimental to the quality of recombinant glycoproteins . Bioreactor temperature has been used to control cell growth and recombinant protein production rates . However, the effect of culture temperature on the production of proteases and sialidases has not been investigated . In this study, Chinese hamster ovary cells were cultured with a temperature profile that decreased from 37 to 34 degrees C over 8 days and with a constant temperature of 37 degrees C . Analysis of extracellular protease activity indicated that two major proteases were present (50 and 69 kDa) . The 50 kDa protease activity decreased similarly with time for both culture conditions . The 69 kDa protease activity increased with time for both culture conditions . The constant-temperature cultures had significantly lower 69 kDa protease levels compared to the ramped-temperature cultures in the early stationary phase . Intracellular sialidase activity was present in both cultures . The intracellular sialidase activity increased dramatically for both culture conditions immediately after the cells were inoculated into fresh medium . The initial peak in intracellular sialidase activity was followed by a first-order decay . The intracellular sialidase activities for the two culture conditions were not significantly different . The production of recombinant tissue type plasminogen activator was not significantly different for the two culture conditions. a, h, c. Thus, the previously hypothesized advantages that lower culture temperatures have reduced protease activity and improved productivity do not appear to be universal.Biotechnol Prog, 2004 Nov-Dec, 20(6), 1802 - 9A novel rotating-shaft bioreactor for two-phase cultivation of tissue-engineered cartilage; Chen HC et al.; A novel rotating-shaft bioreactor (RSB) was developed for two-phase cultivation of tissue-engineered cartilage . The reactor consisted of a rotating shaft on which the chondrocyte/scaffold constructs (7.5 mm diameter x 3.5 mm thickness) were fixed and a reactor vessel half-filled with medium . The horizontal rotation of the shaft resulted in alternating exposure of the constructs to gas and liquid phases, thus leading to efficient oxygen and nutrient transfer, as well as periodically changing, mild shear stress exerting on the construct surfaces (0-0.32 dyn/cm2 at 10 rpm), as revealed by computer simulation . Strategic operation of the RSB (maintaining rotating speed at 10 rpm for 3 weeks and lowering the speed to 2 rpm in week 4) in combination with higher seeding density (6 x 10(6) chondrocytes/scaffold) and medium perfusion resulted in uniform cell distribution and increased glycosaminoglycan (3.1 mg/scaffold) and collagen (7.0 mg/scaffold) deposition . The 4-week constructs resembled native cartilages in terms of not only gross appearance and cell morphology but also distributions of glycosaminoglycan, total collagen, and type II collagen, confirming the maintenance of chondrocyte phenotype and formation of cartilage-like constructs in the RSB cultures . In summary, the novel RSB may be implicated for in vitro study of chondrogenesis and de novo cartilage development under periodic mechanical loading . With proper optimization of the culture conditions, a RSB may be employed for the production of cartilage-like constructs.Biotechnol Prog, 2004 Nov-Dec, 20(6), 1788 - 96High-level scu-PA production by butyrate-treated serum-free culture of recombinant CHO cell line; Kim JS et al.; The MGpUK-5 cell line, transformed with a single-chain urokinase-type plasminogen activator (scu-PA) minigene, generated mRNA transcripts and scu-PA titers corresponding to 65% or 86% of the amount generated before serum-free adaptation, despite significant loss of scu-PA gene copies during adaptation to serum-free culture . To further augment scu-PA production, a culture strategy employing sodium butyrate was explored . In 60-mL spinner flask cultures, sodium butyrate in the concentration range 1-10 mM allowed scu-PA production 2- to 3-fold higher than that in the negative control culture . Its productivity-enhancing activity was dependent on cell density in a range of 1-5 x 10(6) cells/mL, generating 72,200 +/- 8,100 IU/mL (480 +/- 50 mg/L) in 60-mL spinner flask cultures . To confirm this result, cells were grown to 4.4 x 10(6) cells/mL and treated with 5 mM sodium butyrate in a 2.5-L perfusion culture . The scu-PA titer increased more than 2-fold, and specific production rate of scu-PA increased 3-fold by this treatment . Overall, this perfusion culture gave rise to 1.7 x 10(8) IU scu-PA (1.1 g), comparable to total scu-PA production in a batch butyrate-treated culture performed at a 25-L bioreactor scale (1.3-3.5 g) . Our results suggest that sodium butyrate treatment on high-density culture enables scu-PA production in gram quantities.Biotechnol Prog, 2004 Nov-Dec, 20(6), 1725 - 32Polymer development for enhanced delivery of phenol in a solid-liquid two-phase partitioning bioreactor; Prpich GP et al.; Two-Phase Partitioning Bioreactors (TPPBs) have traditionally been used to partition toxic concentrations of xenobiotics from a cell-containing aqueous phase by means of an immiscible organic solvent and to deliver these substrates back to the cells on the basis of metabolic demand and the maintenance of thermodynamic equilibrium between the phases . A limitation of TPPBs, which use organic liquid solvents, is the possibility that the solvent can be bioavailable, and this has therefore limited organic liquid TPPBs to the use of pure strains of microbes . Solid polymer beads have recently been introduced as a replacement for liquid organic solvents, offering similar absorption properties but with the capability to be used with mixed microbial populations . The present work was aimed at identifying a polymer with a greater capacity for and more rapid uptake and release of phenol for use as the second phase in a mixed culture TPPB . Polarity and hydrogen bonding capabilities between polymer and phenol were considered in the screening and selection process of candidate polymers . Hytrel (a copolymer of poly(butylene terephthalate) and butylene ether glycol terephthalate) polymer beads, offered improved capacity (19 mg phenol/g polymer at a fixed initial phenol concentration of 2000 mg/L) and a greater diffusivity (1.54 x 10(-7) cm2/s) when compared to the capacity and diffusivity of previously used EVA (ethylene vinyl acetate) beads (12.4 mg phenol/g polymer and 3.73 x 10(-9) cm2/s, respectively) . Hytrel polymer beads were then used in a TPPB for the investigation of various substrate feeding strategies (fed-batch, bead replacement, and concentrated spikes of phenol), with rapid and complete phenol degradation shown in all cases.Biotechnol Prog, 2004 Nov-Dec, 20(6), 1718 - 24A novel parallel shaken bioreactor system for continuous operation; Akgun A et al.; A novel continuous bioreactor system was developed as a shaken culture vessel for the investigation of the growth kinetics and product formation of microorganisms in milliscale . The novel bioreactor system mainly consists of a specially designed 250-mL shake flask with two inlets, one for gas supply and one for medium supply, and one combined outlet on the side of flask for exhaust gas and culture liquid . As a result of the circulating motion of the fermentation broth in the shake flask, the maximum liquid height reaches the edge of the outlet and the fermentation broth is accelerated into the outlet by centrifugal force . Additionally, the excess fermentation broth leaving the culture vessel is continuously driven by the exhaust gas . Because of the small scale and the simple handling it is possible to operate many of these shaken bioreactor vessels simultaneously . By using parallel vessels operated at different dilution rates on the same shaker, the data for a complete biomass over dilution rate (X-D) diagram of a biological culture can be evaluated in an efficient manner, thus saving money, materials, and time . Continuous fermentations of the yeast Saccharomyces cerevisiae H1022 (ATCC 32167) in the shaken bioreactor system and in a conventional stirred tank fermentor showed very similar results.Biotechnol Prog, 2004 Nov-Dec, 20(6), 1710 - 7Experimental study of a ceramic microsparging aeration system in a pilot-scale animal cell culture; Nehring D et al.; The oxygen supply of cell cultures with the aid of free gas bubbles is an efficient process strategy in pharmaceutical production . If the cell-damaging impact of gas bubbles is reduced, direct aeration becomes a practical solution with scale-up potential and comparatively high oxygen transfer rates . In this paper a microsparging aeration system made of porous ceramic was compared with bubble-free membrane aeration . The sparging system was used for the long-term cultivation of mammalian cells in 2- to 100-L scale bioreactors and produced bubble sizes of 100-500 microm in diameter . Using a scale of 2.5 and 30 L, a cell density of 2.6 x 10(6) cells/mL was attained . When a 100-L scale was used, a density of 1.1 x 10(6) cells/mL was achieved, whereas a comparable membrane-aerated system showed a cell density of 2.2 x 10(6) cells/mL . At relatively low agitation rates of less than 70 rpm in the sparged bioreactors, a homogeneous and constant oxygen concentration was kept in the medium . As a result of the different foam-forming tendency caused by the lower gas flow of the ceramic sparger compared to that of the standard aeration systems, we were able to develop an appropriate process control strategy . Furthermore, oxygen transfer measurements for the common stainless steel sparger and the ceramic sparger showed a 3-fold higher oxygen transfer coefficient for the ceramic sparger.Aviakosm Ekolog Med, 2000, 34(5), 51 - 9{Transmission and exchange of genetic information during bacterial conjugation in ground-based simulations of the factors of orbital flight}; Zerov IuP et al.; Control laboratory experiments on bacterial conjugation under simulated spaceflight conditions were performed with the use of new equipment (bioreactor RECOMB-2 and container BIOMAGNISTAT) within the RSA-NASA science program . External parameters were selected and the plan of simulation of a space experiment was verified to ensure high efficiency of the conjugative transfer of chromosomal and plasmid DNA and storage of hybrids on the ground . Genetic analysis of conjugative hybrids E . coli supported the hypothesized possibility of transfer of a whole bacterial chromosome during conjugation that will lead to relative stabilization of the diploid state . Earlier this hypothesis was used to interpret results of experiments performed on MIR in 1992-1993 . Hence, the ground laboratory investigations proved the conclusion about high probability of transfer of large fragments or even a whole chromosome during space flight . Screening of the geomagnetic field by BIOMAGNISTAT increases the probability of conjugative contacts between cells and is likely to slightly inhibit the processes of recombination.ASAIO J, 2002 Jan-Feb, 48(1), 12 - 6Application of stereolithography for scaffold fabrication for tissue engineered heart valves; Sodian R et al.; A crucial factor in tissue engineering of heart valves is the functional and physiologic scaffold design . In our current experiment, we describe a new fabrication technique for heart valve scaffolds, derived from x-ray computed tomography data linked to the rapid prototyping technique of stereolithography . To recreate the complex anatomic structure of a human pulmonary and aortic homograft, we have used stereolithographic models derived from x-ray computed tomography and specific software (CP, Aachen, Germany) . These stereolithographic models were used to generate biocompatible and biodegradable heart valve scaffolds by a thermal processing technique . The scaffold forming polymer was a thermoplastic elastomer, a poly-4-hydroxybutyrate (P4HB) and a polyhydroxyoctanoate (PHOH) (Tepha, Inc., Cambridge, MA) . We fabricated one human aortic root scaffold and one pulmonary heart valve scaffold . Analysis of the heart valve included functional testing in a pulsatile bioreactor under subphysiological and supraphysiological flow and pressure conditions . Using stereolithography, we were able to fabricate plastic models with accurate anatomy of a human valvular homograft . Moreover, we fabricated heart valve scaffolds with a physiologic valve design, which included the sinus of Valsalva, and that resembled our reconstructed aortic root and pulmonary valve . One advantage of P4HB and PHOH was the ability to mold a complete trileaflet heart valve scaffold from a stereolithographic model without the need for suturing . The heart valves were tested in a pulsatile bioreactor, and it was noted that the leaflets opened and closed synchronously under subphysiological and supraphysiological flow conditions . Our preliminary results suggest that the reproduction of complex anatomic structures by rapid prototyping techniques may be useful to fabricate custom made polymeric scaffolds for the tissue engineering of heart valves.Space Med Med Eng (Beijing), 2001 Apr, 14(2), 149 - 53{Prospect of the Advanced Life Support Program Breadboard Project at Kennedy Space Center in USA}; Guo SS et al.; The Breadboard Project at Kennedy Space Center in NASA of USA was focused on the development of the bioregenerative life support components, crop plants for water, air, and food production and bioreactors for recycling of wastes . The keystone of the Breadboard Project was the Biomass Production Chamber (BPC), which was supported by 15 environmentally controlled chambers and several laboratory facilities holding a total area of 2150 m2 . In supporting the Advanced Life Support Program (ALS Program), the Project utilizes these facilities for large-scale testing of components and development of required technologies for human-rated test-beds at Johnson Space Center in NASA, in order to enable a Lunar and a Mars mission finally.Cell Motil Cytoskeleton, 2001 Nov, 50(3), 161 - 72Saturable binding of the echinoderm microtubule-associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments; Eichenmuller B et al.; The echinoderm microtubule-associated protein (EMAP) is a 75-kDa, WD-repeat protein associated with the mitotic spindle apparatus . To understand EMAP's biological role, it is important to determine its affinity for microtubules (MTs) and other cytoskeletal components . To accomplish this goal, we utilized a low-cost, bubble-column bioreactor to express EMAP as a hexahistidine fusion (6his) protein in baculovirus-infected insect cells . After optimizing cell growth conditions, up to 30 mg of EMAP was obtained in the soluble cell lysate from a 1-liter culture . EMAP was purified to homogeneity in a two-step process that included immobilized metal-affinity chromatography (IMAC) and anion-exchange chromatography . In vitro binding studies on cytoskeletal components were performed with the 6his-EMAP . EMAP bound to MTs, but not actin or vimentin filaments, with an intrinsic dissociation constant of 0.18 microM and binding stoichiometry of 0.7 mol EMAP per mol tubulin heterodimer . In addition, we show that a strong MT binding domain resides in the 137 amino acid, NH(2)-terminus of EMAP and a weaker binding site in the WD-domain . Previous work has shown that the EMAP concentration in the sea urchin egg is over 4 microM . Together, these results show that there is sufficient EMAP in the egg to regulate the assembly of a large pool of maternally stored tubulin . Biotechnol Bioeng, 2002 Mar 20, 77(6), 704 - 16Inhibiting apoptosis in mammalian cell culture using the caspase inhibitor XIAP and deletion mutants; Sauerwald TM et al.; Lower yields and poorer quality of biopharmaceutical products result from cell death in bioreactors . Such cell death may occur from necrosis but is more commonly associated with apoptosis . During the process of programmed cell death or apoptosis, caspases become activated and cause a cascade of events that eventually destroy the cell . XIAP is the most potent caspase inhibitor encoded in the mammalian genome . The effectiveness of XIAP and its deletion mutants was examined in two cell lines commonly utilized in commercial bioreactors: Chinese hamster ovary (CHO) and 293 human embryonic kidney (293 HEK) cells . CHO cells undergo apoptosis as a result of various insults, including Sindbis virus infection and serum deprivation . In this study, we demonstrate that 293 HEK cells undergo apoptosis during Sindbis virus infection and exposure to the toxins, etoposide and cisplatin . Two deletion mutants of XIAP were created; one containing three tandem baculovirus iap repeat (BIR) domains and the other containing only the C-terminal RING domain, lacking the BIRs . Viability studies were performed for cells expressing each mutant and the wild-type protein on transiently transfected cells, as stable pools, or as stable clonal cell populations after induction of apoptosis by serum deprivation, Sindbis virus infection, etoposide, and cisplatin treatment . Expression of the wild-type XIAP inhibited apoptosis significantly; however, the XIAP mutant containing the three BIRs provided equivalent or improved levels of apoptosis inhibition in all cases . Expression of the RING domain offered no protection and was pro-apoptotic in transient expression experiments . With the aid of an N-terminal YFP fusion to each protein, distribution within the cell was visualized, and the wild-type and mutants showed differing intracellular accumulation patterns . While the wild-type XIAP protein accumulated primarily in aggregates in the cytosol, the RING mutant was enriched in the nucleus . In contrast, the deletion mutant containing the three BIRs was distributed evenly throughout the cytosol . Thus, protein engineering of the XIAP protein can be used to alter the intracellular distribution pattern and improve the ability of this caspase inhibitor to protect against apoptosis for two mammalian cell lines . Biotechnol Bioeng, 2002 Mar 20, 77(6), 685 - 92Effect of genetic background and culture conditions on the production of herpesvirus-based gene therapy vectors; Ozuer A et al.; Herpes simplex virus type-1 (HSV-1) represents a unique vehicle for the introduction of foreign DNA to cells of a variety of tissues . The nature of the vector, the cell line used for propagation of the vector, and the culture conditions will significantly impact vector yield . An ideal vector should be deficient in genes essential for replication as well as those that contribute to its cytotoxicity . Advances in the engineering of replication-defective HSV-1 vectors to reduce vector-associated cytotoxicity and attain sustained expression of target genes make HSV-1 an attractive gene-delivery vehicle . However, the yield of the less-cytotoxic vectors produced in standard tissue-culture systems is at least three order of magnitudes lower than that achieved with wild-type virus . The low overall yield and the complexity involved in the preparation of HSV vectors at high concentrations represent major obstacles in use of replication-defective HSV-derived vectors in gene therapy applications . In this work, the dependence of the vector yield on the genetic background of the virus is examined . In addition, we investigated the production of the least toxic, lowest-yield vector in a CellCube bioreactor system . After initial optimization of the operational parameters of the cellcube system, we were able to attain virus yields similar to or better than those values attained using the tissue culture flask system for vector production with significant savings with respect to time, labor, and cost . Environ Toxicol Chem, 2002 Jan, 21(1), 1 - 8Alpha,beta-unsaturated sulfophenylcarboxylates as degradation intermediates of linear alkylbenzenesulfonates: evidence for omega-oxygenation followed by beta-oxidations by liquid chromatography-mass spectrometry; Eichhorn P et al.; Liquid chromatography with an electrospray interface to a mass spectrometer (LC-ES-MS) and LC-ES coupled to a tandem MS (LC-ES-MS/MS) were used to detect and identify intermediates excreted transiently during the aerobic degradation of linear alkylbenzenesulfonates (LAS) in fixed-bed bioreactors (FBBR) . The inoculum for the FBBR was the microflora of the River Rhine, Germany . Two major phenomena were observed on the addition of 100 mg/L LAS to the system, sorption and then biodegradation . Disappearance due to sorption was followed in an inhibited FBBR . Biodegradation of LAS started on day 7 and was accompanied by the transient excretion of intermediates, which were later largely degraded . We detected not only the sulfophenylcarboxylates (SPCs) observed previously but also the alpha,beta-unsaturated SPCs (SPC-2H), which have not been reported before . Experiments with the (4-sulfophenyl)dodecanes (C12-LAS), which had minor contaminants of C11-LAS, showed C12-, C10-, C8-, C6-, and C4-SPCs when LAS was degraded as well as traces of C9-, C7-, and C5-SPCs . Signals from the SPC-2H species were usually some 10% of those from the corresponding SPCs . Samples from these experiments were also examined by gas chromatography-mass spectrometry (GC-MS), but no desulfonated intermediates were detected . We interpret the data to mean that the only attack on LAS was by (omega-oxygenation; there was no visible initial desulfonation . The products of omega-oxygenation were oxidized to the corresponding SPC and subject to beta-oxidation, as evidenced not only by the pattern of C-2 units in the excreted SPCs but also in the corresponding series of SPC-2H, representing the intermediates in beta-oxidation.Crit Rev Biotechnol, 2001, 21(4), 233 - 94Hydrodynamic and mass transfer characteristics of three-phase gaslift bioreactor systems; Petersen EE et al.; This review focuses on the hydrodynamic and mass transfer characteristics of various three-phase, gaslift fluidized bioreactors . The factors affecting the mixing and volumetric mass transfer coefficient (k(L)a), such as liquid properties, solid particle properties, liquid circulation velocity, superficial gas velocity, bioreactor geometry, are reviewed and discussed . Measurement methods, modeling and empirical correlations are reviewed and compared . To the authors' knowledge, there is no 'generalized' correlation to calculate the volumetric mass transfer coefficient, instead, only 'type-specific' correlations are available in the literature . This is due to the difficulty in modeling the gaslift bioreactor, caused by the variation in geometry, fluid dynamics, and phase interactions . The most important design parameters reported in the literature are: gas hold-up, liquid circulation velocity, 'true' superficial gas velocity, mixing, shear rate, aeration rate and volumetric mass transfer coefficient, k(L)a.Int J Artif Organs, 2001 Nov, 24(11), 807 - 13The effect of serum from liver cancer patients on the growth and function of primary and immortalised hepatocytes; Grant MH et al.; A limiting factor in the efficacy of bioartificial liver (BAL) for the treatment of liver failure is the toxicity of the patients' serum to the hepatocytes in the device . This study investigates the interaction of liver cancer patient serum with primary and immortalised rat hepatocytes . Liver cancer serum increased the growth rate of immortalised hepatocytes, without affecting reduced glutathione levels . The activities of DT-diaphorase and pi glutathione-S-transferase (GST), enzymes associated with de-differentiation, were also increased . Exposure of primary hepatocytes to liver cancer serum resulted in a decrease in cytochrome P450 (CYP) content, and in P450 dependent metabolism of testosterone . Formation of 2-alpha- and 6-beta- hydroxy testosterone was decreased . These reactions are predominantly associated with CYP 2C11 and 3A1 respectively in normal rat liver . The activity of total GST was also decreased, although that of the pi isoenzyme of GST was not affected . Our results suggest that exposure of hepatocytes in a bioreactor to liver cancer patient serum will result in overgrowth of cells, if proliferating cells are being used, and in de-differentiation . The serum may have to be pretreated with adsorbants to remove toxins prior to BAL treatment.Int J Artif Organs, 2001 Nov, 24(11), 793 - 8Experimental evaluation of a cell module for hybrid liver support; Gerlach JC et al.; Aim of the study was to evaluate a hybrid liver support system in a porcine model of acute liver failure, after hepatectomy . Pigs with a body weight of 70+/-18 kg underwent total hepatectomy and porto-cavo-caval shunting as well as ligation of the bile duct and the hepatic artery . Control animals were connected to the system (including capillary membrane plasma separation) containing a four compartment bioreactor with integral oxygenation and decentralized mass exchange but without liver cells . The treatment group received hybrid liver support with the same system including 370+/-42 g primary isolated porcine parenchymal liver cells in co-culture with hepatocyte nursing cells, tissue engineered to liver- like structures at high density . Treatment started after complete recovery from anesthesia and was performed continuously . A positive influence on peripheral vascular resistance and a reduced need of catecholamine dosage was observed in the treatment group . Hybrid liver support with a cell module upscaled for clinical application significantly prolonged survival time in animals after hepatectomy with the longest survival being 26 hours in the control group an 57 hours in the treatment group.Ann N Y Acad Sci, 2001 Nov, 944, 334 - 43Effect of flow configuration and membrane characteristics on membrane fouling in a novel multicoaxial hollow-fiber bioartificial liver; MacDonald JM et al.; A novel "multicoaxial hollow fiber bioreactor" has been developed consisting of four concentric tubes, the two innermost tubes are called hollow fibers . Bioartificial livers are created by culturing liver progenitors in the space between the two innermost hollow fibers and with culture media contained in the two compartments (intracapillary and extracapillary) sandwiching the cell compartment . The outermost compartment is used for gas exchange . A hydrodynamic model has recently been established to predict the optimum hydraulic permeability and bioreactor operational parameters to create the physicochemical environment found in the liver acinus . However, perfusion with serum-free hormonally-defined media and inoculation of cells introduces membrane fouling into the equation, and this parameter must be incorporated into the model . Using commercially available semipermeable hollow fibers (1 mm {0.65 microm pores} and 3 mm {0.1 microm pores} outer diameters {o.d}), the primary cause of resistance is the middle hollow fiber . Preliminary studies using bioreactors inoculated with isolated rat hepatocytes and perfused with serum-containing culture media demonstrated that the middle hollow fiber is the primary site of fouling, and this fouling ultimately causes cell mortality by blocking the transfer of nutrients . Experiments were performed to determine the best commercially available middle hollow fiber for construction of bioreactors and two 3-mm outer-diameter middle hollow fibers were compared: polypropylene and polysulfone, with 0.2 microm and 0.1 microm pore sizes, respectively . Dead-ended and cross flow configurations were compared for their effectiveness at reducing membrane fouling in the middle hollow fiber by determining the change in resistance with time . The results demonstrate that the 0.2-microm pore size polypropylene hollow fiber is the best choice for construction of the multicoaxial hollow-fiber bioreactor, and that cross flow results in two orders of magnitude lower resistance than dead-ended flow after 36 h.Ann N Y Acad Sci, 2001 Nov, 944, 308 - 19Development of a hybrid liver support system; Sauer IM et al.; Hybrid liver systems are being developed as temporary extracorporeal liver support therapy . The overview given here emphasizes the development of both hepatocyte culture models for bioreactors and of systems for clinical therapy . In vitro studies demonstrate long term external metabolic function in isolated primary hepatocytes within bioreactors . These systems are capable of supporting essential liver functions . Animal experiments verify the possibility of upscaling bioreactors for clinical treatment . However, since there is no reliable animal model for investigating the treatment of acute liver failure, the promising results obtained from these studies have limited relevance to human beings . The small number of clinical studies performed thus far are not sufficient to enable any conclusions concerning improvements in the therapy of acute liver failure . Although important progress has been made in the development of these systems, multiple hepatocyte culture models and bioreactor constructions are being discussed in the literature, indicating competition in this field of medical research . For the use of hepatocytes and sinusoidal endothelial cells in coculture, a bioreactor has been designed . The construction is based on capillaries for hepatocyte aggregate immobilization . 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 . The parallel use of identical units enables easy upscaling . Initial studies on the use of discarded organs that are unsuitable for transplantation as a source for primary human liver cells seem to be promising.Sheng Wu Gong Cheng Xue Bao, 2001 Sep, 17(5), 561 - 5{Studies on the cell suspension culture of Saussarea medusa in a stirred tank bioreactor}; Huang Y et al.; The cell suspension culture of Saussarea medusa in a 2L aerated and agitated bioreactor with a four-pitch-blade impeller was investigated . The effects of agitation speed, aeration and inoculum size on cell growth and flavonoids production were studied and it was found that cells had optimum growth and flavonoids production when cultivated at 75 r/min, 700-1000 L/min and an inoculum of 4.0-5.0 g/L . A high cell biomass of 13.8 g/L and flavonoids production of 416 mg/L were achieved after 12 days of cultivation . Time course study revealed that flavonoids biosynthesis was growth-associated . The studies on aggregates size distribution in the bioreactor showed that the aggregates break-up caused by hydrodynamic stress might adversely affect cell growth and lead to significant reduction of cell biomass and flavonoids production.Water Sci Technol, 2001, 44(10), 197 - 202Sludge reduction potential of metazoa in membrane bioreactors; Luxmy BS et al.; In order to check the sludge reduction capacity of metazoa in a membrane bioreactor (MBR), pilot-scale studies were conducted . Three MBRs had been set in a wastewater treatment plant at Tokyo, Japan and they were receiving real wastewater . Initially pH inside the three MBRs was controlled as pH 7, 6 and 5 respectively . Then metazoa population was monitored along with MLSS change . It was found that the presence or absence of the metazoa population did not have any significant effect on the increasing pattern of MLSS . In the MBR with pH 6 highest accumulation of sludge was observed though a high and steady level of metazoa (1,000-2,000 per ml) was present there . But in this MBR a lot of metazoa attached in the membrane was also observed and here the increase in transmembrane pressure was less than in the other two . So, metazoa population especially the attached one in the membrane plays an effective role in fouling control of the membrane . Presence of attached media may provide a suitable niche for metazoa in the process . So, attached media known as DB lace was also inserted in MBRs for testing its capacity along with inoculum of oligochaete worms . Accumulation of sludge was not satisfactory in the attached string and it seems that inoculated worm could not adjust to the environment as they were not sludge originated . So, in the next experimental stage, attached media was inserted in the form of a bundle and this time no inoculation of worm was used . A steady metazoa population was observed in the system but the accumulation of sludge in the attached media was the same as before.Ann Biomed Eng, 2001 Nov, 29(11), 963 - 73Dynamic in vitro quantification of bioprosthetic heart valve leaflet motion using structured light projection; Iyengar AKS et al.; Quantification of heart valve leaflet deformation during the cardiac cycle is essential in understanding normal and pathological valvular function, as well as in the design of replacement heart valves . Due to the technical complexities involved, little work to date has been performed on dynamic valve leaflet motion . We have developed a novel experimental method utilizing a noncontacting structured laser-light projection technique to investigate dynamic leaflet motion . Using a simulated circulatory loop, a matrix of 150-200 laser light points were projected over the entire leaflet surface . To obtain unobstructed views of the leaflet surface, a stereo system of high-resolution boroscopes was used to track the light points at discrete temporal points during the cardiac cycle . The leaflet surface at each temporal point was reconstructed in three dimensions, and fit using our biquintic hermite finite element approach (Smith et al., Ann . Biomed . Eng . 26:598-611, 2001) . To demonstrate our approach, we utilized a bovine pericardial bioprosthetic heart valve, which revealed regions of complex flexural deformation and substantially different shapes during the opening and closing phases . In conclusion, the current method has high spatial and temporal resolution and can reconstruct the entire surface of the cusp simultaneously . Because it is completely noncontacting, this approach is applicable to studies of fatigue and bioreactor technology for tissue engineered heart valves.Ann Biomed Eng, 2001 Nov, 29(11), 947 - 55Analysis of oxygen transport to hepatocytes in a flat-plate microchannel bioreactor; Roy P et al.; Oxygen transfer to cultured hepatocytes in microchannel parallel-plate bioreactors with and without an internal membrane oxygenator was investigated with a mathematical model and the results were corroborated with fluorescence imaging experiments . The consumption of oxygen by hepatocytes was assumed to follow Michaelis-Menten kinetics . Our simulations indicate that under conditions of low Peclet (Pe) number (<80)>20 mg/l of purified His-tagged SEAP was recovered from a 3.5 l bioreactor . Intracellular proteins were also produced at levels as high as 50 mg/l, representing up to 20% of total cell proteins.