Biotechnol Prog, 2004 Nov-Dec, 20(6), 1660 - 5 Improved bioconversion of 15-fluoro-6-deoxyerythronolide B to 15-fluoro-erythromycin A by overexpression of the eryK Gene in Saccharopolyspora erythraea; Desai RP et al.; The bioconversion of a 6-deoxyerythronolide B analogue to the corresponding erythromycin A analogue (R-EryA) by a Saccharopolyspora erythraea mutant lacking the ketosynthase in the first polyketide synthase module was significantly improved by changing fluxes at a key branch point affecting the erythromycin congener distribution . This was achieved by integrating an additional copy of the eryK gene into the chromosome under control of the eryAIp promoter . Real-time PCR analysis of RNA confirmed higher expression of eryK in the resulting strain, S . erythraea K301-105B, compared to its parent . In shake flasks, K301-105B produced less of the shunt product 15-fluoro-erythromycin B (15F-EryB), suggesting a shift in congener distribution toward the desired product, 15-fluoro-erythromycin A (15F-EryA) . In bioreactor studies, K301-105B produced 1.3 g/L of 15F-EryA with 75-80% molar yield on fed precursor, compared with 0.9 g/L 15F-EryA with 50-55% molar yield on fed precursor by the parent strain . At higher precursor feed rates, K301-105B produced 3.5 g/L of 15F-EryA while maintaining 75-80% molar yield on fed precursor. Biotechnol Prog, 2004 Nov-Dec, 20(6), 1634 - 40 A high-throughput approach to promoter study using green fluorescent protein; Lu C et al.; Green fluorescent protein (GFP) is a reporter that has had a significant impact due to its many advantages over other reporter genes: it is autofluorescent, it enables in situ detection, it is relatively small, and it is also nontoxic . By cloning a gene promoter upstream of the gfp gene and exposing the living cells transformed with the fusion to the specific inducer or repressor, gene expression can be real-time monitored by continuous quantitative measurement of the green fluorescence emitted by GFP . In this work, a promoter study using promoter-gfp fusions was conducted in 96-well plates . Because they were placed in an automated incubating and shaking microplate reader, the wells functioned as microscale bioreactors, allowing for parallel experiments and data analysis . In the study described here, an overexpression promoter (pBAD promoter) and two comparatively weak promoters (sodA and acnA in Escherichia coli SoxRS regulon) were studied in both endpoint and kinetics formats . Our results with the pBAD promoter revealed insight on its regulation, which is tightly controlled by levels of arabinose and glucose . Results on weak oxidative stress promoters (for sodA and acnA genes) were striking in that significant induction was observed when they were under a superoxide stress in plates . They both displayed dose-dependent induction to paraquat-generated superoxide anion, with sodA leading acnA in strength and time . These results, spanning highly inducible promoters for protein overexpression and weakly inducible promoters of metabolic interest, demonstrate that the approach is relatively easily executed and can be used for quantitative and temporal promoter studies in a high throughput format. Brain Res Bull, 2004 Dec 15, 64(4), 347 - 56 Rapid differentiation of NT2 cells in Sertoli-NT2 cell tissue constructs grown in the rotating wall bioreactor; Saporta S et al.; Cell replacement therapy is of great interest as a long-term treatment of neurodegenerative diseases such as Parkinson's disease (PD) . We have previously shown that Sertoli cells (SC) provide neurotrophic support to transplants of dopaminergic fetal neurons and NT2N neurons, derived from the human clonal precursors cell line NTera2/D1 (NT2), which differentiate into dopaminergic NT2N neurons when exposed to retinoic acid . We have created SC-NT2 cell tissue constructs cultured in the high aspect ratio vessel (HARV) rotating wall bioreactor . Sertoli cells, NT2, and SC plus NT2 cells combined in starting ratios of 1:1, 1:2, 1:4 and 1:8 were cultured in the HARV in DMEM with 10% fetal bovine serum and 1% growth factor reduced Matrigel for 3 days, without retinoic acid . Conventional, non-HARV, cultures grown in the same culture medium were used as controls . The presence of tyrosine hydroxylase (TH) was assessed in all culture conditions . Sertoli-neuron-aggregated-cell (SNAC) tissue constructs grown at starting ratios of 1:1 to 1:4 contained a significant amount of TH after 3 days of culture in the HARV . No TH was detected in SC HARV cultures, or SC, NT2 or SC-NT2 conventional co-cultures . Quantitative stereology of immunolabled 1:4 SNAC revealed that approximately 9% of NT2 cells differentiate into TH-positive (TH+) NT2N neurons after 3 days of culture in the HARV, without retinoic acid . SNAC tissue constructs also released dopamine (DA) when stimulated with KCl, suggesting that TH-positive NT2N neurons in the SNAC adopted a functional dopaminergic phenotype . SNAC tissue constructs may be an important source of dopaminergic neurons for neuronal transplantation. J Environ Sci (China), 2004, 16(5), 874 - 80 Comparison between controlled landfill reactor and conditioned landfill bioreactor; Luo F et al.; Bioreactor landfills allow a more active landfill management that recognizes the biological, chemical and physical processes involved in a landfill environment . The laboratory-scale simulators of landfill reactors treating municipal solid wastes were studied, the effect of solid waste size, leachate recirculation, nutrient balance, pH value, moisture content and temperature on the rate of municipal solid waste (MSW) biodegradation were determined, and it indicated the optimum pH value, moisture content and temperature decomposing MSW . The results of waste biodegradation were compared with that of the leachate-recirculated landfill simulator and conservative sanitary landfill simulator . In the control experiment the antitheses of a decreasing trend of the organic load, measured as biological oxygen demand and chemical oxygen demand, was shown . An obvious enhancement of effective disposal from conservative sanitary landfill (CSL) simulator, to the leachate-recirculated landfill (LRL) simulator and to the conditioned bioreactor landfill (CBL) simulator would be noted, through displaying the compared results of solid waste settlement, heavy metal concentration in leachate, methane production rate, biogas composition, BOD and COD as well as their ratio. Orthopade . 2004 Nov 19; {Epub ahead of print} {Tissue engineering of long bones with a vascular matrix in a bioreactor.}; Jagodzinski M et al.; BACKGROUND . The purpose of this study was to assess the feasibility of tissue engineering a vascular osteogenic construct . METHODS . The capillary system of a vascular biologic matrix (BioVaM) was seeded with human bone marrow stromal cells (hBMSC) that were differentiated in an endothelial cell-specific media . Within two layers of the vascular matrix, hBMSC were either cultivated with demineralized bone matrix (DBM) or liquefied small intestine submucosa (SIS) . After 3 and 6 weeks in a perfusion bioreactor, endothelial cell phenotype and cell adherence were investigated with immunohistology . The contents of alkaline phosphatase (ALP) and osteocalcin (OC) were determined and a load to failure test was executed . Histologic differences were analyzed using H&E and von Kossa staining . RESULTS . The capillary system of the matrix stained positive for endothelial markers . ALP activity increased in DBM after 3 and 6 weeks (2.2+/-1.0 and 2.4+/-0.6 U/l/microg protein, p<0.05) . Osteocalcin levels were highest for DBM (23.5+/-21.1 and 11.1+/-4.5 ng/ml, p<0.05) . Tissue strength was enhanced in SIS and DBM after 6 weeks (15.1+/-3.7 N/mm(2) and 17.0+/-5.0 N/mm(2), p<0.05) . Tissue morphology resembled osteons in DBM and SIS cultures; however, areas that stained positive for phosphate were more often found in the DBM group . CONCLUSION . The capillary system of the matrix could be seeded with BMSC in vitro . Seeding of hBMSC mixed with DBM or SIS within a vascular matrix led to enhanced tissue strength and to osteon-like tissue structures . Osteogenic differentiation was highest when DBM was used. Biomed Microdevices, 2004 Dec, 6(4), 325 - 39 NanoLiterBioReactor: long-term mammalian cell culture at nanofabricated scale; Prokop A et al.; There is a need for microminiaturized cell-culture environments, i.e . NanoLiter BioReactors (NBRs), for growing and maintaining populations of up to several hundred cultured mammalian cells in volumes three orders of magnitude smaller than those contained in standard multi-well screening plates . These devices would enable the development of a new class of miniature, automated cell-based bioanalysis arrays for monitoring the immediate environment of multiple cell lines and assessing the effects of drug or toxin exposure.We fabricated NBR prototypes, each of which incorporates a culture chamber, inlet and outlet ports, and connecting microfluidic conduits . The fluidic components were molded in polydimethylsiloxane (PDMS) using soft-lithography techniques, and sealed via plasma activation against a glass slide, which served as the primary culture substrate in the NBR . The input and outlet ports were punched into the PDMS block, and enabled the supply and withdrawal of culture medium into/from the culture chamber (10-100 nL volume), as well as cell seeding . Because of the intrinsically high oxygen permeability of the PDMS material, no additional CO(2)/air supply was necessary.The developmental process for the NBR typically employed several iterations of the following steps: Conceptual design, mask generation, photolithography, soft lithography, and proof-of-concept culture assay . We have arrived at several intermediate designs . One is termed "circular NBR with a central post (CP-NBR)," another, "perfusion (grid) NBR (PG-NBR)," and a third version, "multitrap (cage) NBR (MT-NBR)," the last two providing total cell retention.Three cells lines were tested in detail: a fibroblast cell line, CHO cells, and hepatocytes . Prior to the culturing trials, extensive biocompatibility tests were performed on all materials to be employed in the NBR design . To delineate the effect of cell seeding density on cell viability and survival, we conducted separate plating experiments using standard culture protocols in well-plate dishes . In both experiments, PicoGreen assays were used to evaluate the extent of cell growth achieved in 1-5 days following the seeding . Low seeding densities resulted in the absence of cell proliferation for some cell lines because of the deficiency of cell-cell and extracellular matrix (ECM)-cell contacts . High viabilities were achieved in all designs.We conclude that an instrumented microfluidics-based NanoBioReactor (NBR) will represent a dramatic departure from the standard culture environment . The employment of NBRs for mammalian cell culture opens a new paradigm of cell biology, so far largely neglected in the literature. Philos Transact A Math Phys Eng Sci, 2004 Dec 15, 362(1825), 2821 - 50 Bone repair in the twenty-first century: biology, chemistry or engineering? Hing KA. Increases in reconstructive orthopaedic surgery, such as total hip replacement and spinal fusion, resulting from advances in surgical practice and the ageing population, have lead to a demand for bone graft that far exceeds supply . Consequently, a number of synthetic bone-graft substitutes (BGSs) have been developed with mixed success and surgical acceptance . Skeletal tissue regeneration requires the interaction of three basic elements: cells, growth factors (GFs) and a permissive scaffold . This can be achieved by pre-loading a synthetic scaffold with GFs or pre-expanded cells; however, a 'simpler' approach is to design intrinsic 'osteoinductivity' into your BGS, i.e . the capability to recruit and stimulate the patient's own GFs and stem cells . Through investigation of the mechanisms controlling bone repair in BGSs, linking interactions between the local chemical and physical environment, scientists are currently developing osteoinductive materials that can stimulate bone regeneration through control of the scaffold chemistry and structure . Moreover, this body of research is providing the foundations for future generations of BGSs and bone-repair therapies and may ultimately contribute towards improving the quality of life through maintenance of the skeleton and reversal of disease states, as opposed to the mending of broken bones that we currently practice . Will we be able to grow our own bones in a bioreactor for use as autologous graft materials in the future? Could surgery be limited to accidental trauma cases, with greater restoration of function through biochemical or gene therapies? The technology and research probes necessary to this task are currently being developed with the advent of nanotechnology, genomics and proteomics: are we about to embark on a chemical revolution in medicine? This paper aims to discuss some of the current thinking on the mechanisms behind bioactivity and biocompatibility in bone and how a fuller understanding of the interactions between cells and the materials used today could bring about completely new approaches for the treatment of bone fracture and disease tomorrow. Biotechnol Bioeng, 2004 Dec 30, 88(7), 854 - 60 Physical and hydrodynamic properties of flocs produced during biological hydrogen production; Zhang JJ et al.; Dense flocs readily form in continuous culture bioreactors used for hydrogen production, but the fractal and hydrodynamic properties of these flocs have not been previously analyzed . We therefore examined the size distribution, fractal dimension, and hydrodynamic properties of flocs formed in a continuous flow, well-mixed reactor treating synthetic wastewater at a fixed condition of a 4.5 h hydraulic detention time (23 degrees C, pH 5.5) . The reactor was operated for a total of 3 months at three different organic loading rates (27, 53, and 80 g-COD/L-d) with influent glucose concentrations of 5, 10, and 15 g-COD/L . At all three loading rates the removal of glucose was nearly complete (98.6-99.4%) and biomass was produced in proportion to the organic loading rate (0.86 +/- 0.11, 2.40 +/- 0.26, and 4.59 +/- 1.55 g/L of MLVSS in the reactor) . Overall conversion efficiencies of glucose to hydrogen, evaluated on the basis of a maximum of 4 mol-H2/mol-glucose, increased with organic loading rates in the order 17.7%, 23.1%, and 25.6% . The gas contained 56.1 +/- 4.9% hydrogen, with the balance as carbon dioxide . No methane gas was detected . Under these conditions, flocs were produced with mean sizes that increased with organic loading, in the order 0.12 cm (5 g-COD/L), 0.35 cm (10 g-COD/L), and 0.58 cm (15 g-COD/L) . As the average floc size increased, the flocs became on average denser and less fractal, with fractal dimensions increasing from 2.11 +/- 0.17 to 2.48 +/- 0.13 . Floc porosities ranged from 0.75-0.96, and resulted in aggregate densities that allowed little intra-aggregate flow through the floc . As a result, average settling velocities were not appreciably larger than those predicted by Stokes' law for spherical, impermeable flocs . Our results demonstrate that dense, relatively impermeable flocs are produced in biohydrogen reactors that have settling properties in reasonable agreement with Stokes' law . 2004 Wiley Periodicals, Inc. J Gene Med, 2004 Dec, 6(12), 1293 - 303 Improved gene transfer and normalized enzyme levels in primitive hematopoietic progenitors from patients with mucopolysaccharidosis type I using a bioreactor; Pan D et al.; BACKGROUND: One of the major barriers to the clinical application of hematopoietic stem cell (HSC) gene therapy has been relatively low gene transfer efficiency . Other inadequacies of current transduction protocols are related to their multi-step procedures, e.g., using tissue-culture flasks, roller bottles or gas-permeable bags for clinical application . METHODS: In comparison with a conventional bag transduction protocol, a 'closed' hollow-fiber bioreactor system (HBS) was exploited to culture and transduce human peripheral blood CD34(+) progenitor cells (PBPC(MPS)) from patients with mucopolysaccharidosis type I (MPS I) using an amphotropic retroviral vector based on a murine Moloney leukemia virus LN prototype . Both short-term colony-forming cell (CFC) and long-term culture initiating cell (LTCIC) assays were employed to determine transduction frequency and transgene expression in committed progenitor cells and primitive progenitors with multi-lineage potentials . RESULTS: A novel ultrafiltration-transduction method was established to culture and transduce enzyme-deficient PBPC(MPS) over a 5-day period without loss in viability and CD34 identity (n = 5) . Significantly higher transduction efficiencies were achieved in primary CFC that derived from the HBS (5.8-14.2%) in comparison with those from gas-permeable bags (undetectable to 1.7%; p < 0.01) . Up to 15-fold higher-than-normal enzyme activity was found in selected PBPC(MPS)-LP1CD transductants . Moreover, higher gene transfer (4.4-fold) and expression in very primitive progenitors were observed in products from the HBS compared with bag experiments as indicated by CFC derived from primitive LTCIC . Remarkably, with relatively modest gene transfer levels in LTCIC from HBS experiments, the expression of the IDUA transgene corrected the enzyme-deficiency in 5-week long-term cultures (LTC) . CONCLUSIONS: MPS I progenitor cells achieved normalized enzyme levels in LTC after transduction in a HBS system . These studies demonstrate the advantages of a bioreactor-transduction system for viral-mediated stem cell gene transfer . Copyright (c) 2004 John Wiley & Sons, Ltd. J Gene Med, 2004 Dec, 6(12), 1369 - 81 Development and characterization of a cell line for large-scale, serum-free production of recombinant adeno-associated viral vectors; Farson D et al.; BACKGROUND: One of the major limitations to the use of adeno-associated virus (AAV) vectors for gene therapy has been the difficulty in producing enough vector to supply a clinical trial . More than 20 000 roller bottles may be required to generate AAV by the traditional transient transfection process to treat 50 patients . A scalable AAV producer cell line grown in serum-free media will meet the needs for the manufacture of AAV gene therapeutics . METHODS: A packaging cell line was generated by introducing the AAV rep and cap genes into A549 cells . From this packaging cell line, a number of producer cell lines were generated by infecting the packaging cell with the appropriate AAV vector . Producer cell lines were then adapted to serum-free suspension conditions for growth in bioreactors . RESULTS: We report here the development of six AAV producer cell lines that generate > 10(4) particles/cell . The rAAV vector preparations from these cell lines have physical and functional characteristics similar to rAAV vectors prepared by transient transfection . To enable large-scale production, producer cell lines were adapted to serum-free suspension and we demonstrate production of AAV at the 15 L scale . In addition, vector preparations from these cell lines were shown to be free of wild-type AAV . CONCLUSIONS: AAV producer cell lines can be readily scaled to meet the needs of clinical trials . One 500 L bioreactor of these producer cells can produce the equivalent of 2500 high capacity roller bottles or 25 000 T-175 tissue culture flasks . Copyright (c) 2004 John Wiley & Sons, Ltd. J Environ Qual, 2004 Nov-Dec, 33(6), 2026 - 9 Biological reduction of chlorate in a gas-lift reactor using hydrogen as an energy source; Kroon AG et al.; Chlorate release into the environment occurs with its manufacture and use . Biological reduction of chlorate offers an attractive option to decrease this release . A hydrogen gas-lift reactor with microorganisms attached to pumice particles was used for the treatment of wastewater containing high concentrations of chlorate . The microorganisms used chlorate as an electron acceptor and hydrogen gas as a reducing agent . After a start-up period of only a few weeks, chlorate reduction rates of 3.2 mmol L(-1) h(-1) were achieved during continuous operation . During this period, a hydrogen consumption rate of 14.5 mmol L(-1) h(-1) was observed . Complete removal of chlorate was maintained at hydraulic retention times of 6 h . This study clearly demonstrates the potential of hydrogen gas-lift bioreactors for the treatment of chlorate-containing waste streams. J Environ Sci Health A Tox Hazard Subst Environ Eng, 2004, 39(11-12), 3029 - 81 Integration of advanced oxidation technologies and biological processes: recent developments, trends, and advances; Tabrizi GB et al.; The greatest challenge of today's wastewater treatment technology is to optimize the use of biological and chemical wastewater treatment processes . The choice of the process and/or integration of the processes depend strongly on the wastewater characteristics, concentrations, and the desired efficiencies . It has been observed by many investigators that the coupling of a bioreactor and advanced oxidation processes (AOPs) could reduce the final concentrations of the effluent to the desired values . However, optimizing the total cost of the treatment is a challenge, as AOPs are much more expensive than biological processes alone . Therefore, an appropriate design should not only consider the ability of this coupling to reduce the concentration of organic pollutants, but also try to obtain the desired results in a cost effective process . To consider the total cost of the treatment, the residence time in biological and photochemical reactors, the kinetic rates, and the capital and operating costs of the reactors play significant roles . In this study, recent developments and trends (1996-2003) on the integration of photochemical and biological processes for the degradation of problematic pollutants in wastewater have been reviewed . The conditions to get the optimum results from this integration have also been considered . In most of the studies, it has been shown that the integrated processes were more efficient than individual processes . However, slight changes in the configuration of the reactors, temperature, pH, treatment time, concentration of the oxidants, and microorganism's colonies could lead to a great deviation in results . It has also been demonstrated that the treatment cost in both reactors is a function of time, which changes by the flow rate . The minimum cost in the coupling of the processes cannot be achieved unless considering the best treatment time in chemical and biological reactors individually. J Environ Sci Health A Tox Hazard Subst Environ Eng, 2004, 39(11-12), 2955 - 72 Effect of aeration on the performance of a simulated landfilling reactor stabilizing municipal solid wastes; Agdag ON et al.; In this study, the effects of intermittent aeration on the anaerobic treatment of domestic solid waste and leachate characteristics were investigated in three simulated landfill anaerobic bioreactors . All of the reactors were operated with leachate recirculation and recirculation rate was 300 mL/d . All the reactors were loaded with solid waste having different operational mode . The first reactor was operated with recirculation (control-no aeration) . The second reactor was aerated three days in a week during 1 h (Run 1) and the last reactor was aerated one day in a week during 1 h (Run 2) . pH, chemical oxygen demand (COD), volatile fatty acids (VFA), ammonium nitrogen (NH4-N), nitrate nitrogen (NO3-N) concentrations, total, and methane gas productions in the leachate samples were regularly monitored . 96, 85, and 97% COD and 86, 77, and 93% VFA removals were obtained, in the leachate samples of the control, R1 and R2 reactors, respectively, after 79 days of anaerobic incubation . The TN, TP and NH4-N concentrations in MSW reduced to 4300, 1100, and 160 mg/g from 11,100, 2450, and 630 mg/g in R1 reactor by day 79 . The values of pH were 6.98, 6.76, and 7.98, respectively, after anaerobic incubation, respectively in the aforementioned reactors . It was observed that the aeration decreased the methane percentage and the quantity in the simulated bioreactor . The maximum cumulative methane gas production was recorded as 11.2, 0.9, and 3.6 L in control, R1 and R2 reactors, respectively, at the end of 79 days . A BOD5/COD ratio of 0.23 achieved in the R2 reactor indicated the better MSW stabilization resulting in a high rate than that of R1 and control reactors . It was observed that the aerated reactor one day in a week during one hour (R2) reduced the COD and VFA concentrations in leachate samples . The aerated reactor three days in a week during 1 h (R1) reduced the waste quantity, the organic content of the solid waste . The final leachate quantities of aerated runs were lower than that of control reactor operated under strictly anaerobic conditions. Biotechnol Bioeng, 2004 Dec 20, 88(6), 778 - 87 Synthesis of isomaltooligosaccharides and oligodextrans in a recycle membrane bioreactor by the combined use of dextransucrase and dextranase; Goulas AK et al.; A recycle ultrafiltration membrane reactor was used to develop a continuous synthesis process for the production of isomaltooligosaccharides (IMO) from sucrose, using the enzymes dextransucrase and dextranase . A variety of membranes were tested and the parameters affecting reactor stability, productivity, and product molecular weight distribution were investigated . Enzyme inactivation in the reactor was reduced with the use of a non-ionic surfactant but its use had severe adverse effects on the membrane pore size and porosity . During continuous isomaltooligosaccharide synthesis, dextransucrase inactivation was shown to occur as a result of the dextranase activity and it was dependent mainly on the substrate availability in the reactor and the hydrolytic activity of dextranase . Substrate and dextranase concentrations (50-200 mg/mL(-1) and 10-30 U/mL(-1), respectively) affected permeate fluxes, reactor productivity, and product average molecular weight . The oligodextrans and isomaltooligosaccharides formed had molecular weights lower than in batch synthesis reactions but they largely consisted of oligosaccharides with a degree of polymerization (DP) greater than 5, depending on the synthesis conditions . No significant rejection of the sugars formed was shown by the membranes and permeate flux was dependent on tangential flow velocity. Biotechnol Bioeng, 2004 Dec 20, 88(6), 699 - 706 Screening for improved cell performance: selection of subclones with altered production kinetics or improved stability by cell sorting; Bohm E et al.; One of the major problems in the biotechnology industry is the selection of cell lines well suited for production of biopharmaceutical proteins . Usually, the most important selection criterion is the cell specific production rate . Nevertheless, a good producer cell line should have a number of additional advantageous properties, which allow the cell line to perform well in the type of bioreactor chosen for the process . However, the time and work required to select for high production rates as well as the lack of methods to specifically select for other cellular properties, usually prevents researchers from including such criteria into their screening program.With the Single Cell Secretion Assay it is possible to measure the specific production rates of individual cells by catching secreted product in an artificial matrix applied to the cell surface . Flow cytometric cell sorting then allows selection of rare cells with high production rates, which occur at frequencies as low as 10(-6) . By combining this method with culture conditions that bring out a desired cellular property, we were able to isolate subclones with similar production rates, but improved performance from a recombinant Chinese hamster ovary cell line producing a human monoclonal antibody . The two desired cellular properties screened for were a non-growth associated production kinetic and improved stability in the absence of selective pressure. Biotechnol Bioeng, 2004 Dec 30, 88(7), 909 - 15 Characterization of the metabolic burden on Escherichia coli DH1 cells imposed by the presence of a plasmid containing a gene therapy sequence; Rozkov A et al.; The presence of a plasmid, containing gene sequences for DNA immunotherapy that are not expressed in microbial culture, imposed a degradation in bioreactor performance in cultures of the host E . coli strain . Significant decreases in growth rate (24%) and biomass yield (7%) and a corresponding increase in overflow metabolism were observed in a strain containing a therapeutic sequence (a hepatitis B antigen under the control of a CMV promotor) . The observed increase in overflow metabolism was incorporated into a Metabolic Flux Analysis (MFA) model (as acetate secretion) . Metabolic flux analysis revealed an increase in TCA cycle flux, consistent with an increased respiration rate observed in plasmid-containing cells . These effects are thought to result from increased ATP synthesis requirements (24%) arising from the expression of the Kanr plasmid marker gene whose product accounted for 18% of the cell protein of the plasmid-containing strain . These factors will necessitate significantly higher aeration and agitation rates or lower nutrient feed rates in high-density cultures than would be expected for plasmid-free cultures . 2004 Wiley Periodicals, Inc. J Exp Biol, 2004 Nov, 207(Pt 24), 4283 - 9 Rumen metabolites serve ticks to exploit large mammals; Donze G et al.; Hard ticks spend most of their life isolated from passing vertebrates but require a blood meal to proceed to the next life stage (larva, nymph or adult) . These opportunist ectoparasites must be capable of anticipating signals that render suitable hosts apparent . Large ungulates that tolerate a high ectoparasite burden are the favoured hosts of adult hard ticks . Ruminants, comprising the majority of ungulate species, must regularly eruct gases from the foregut to relieve excess pressure and maintain a chemical equilibrium . Through eructations from individuals, and particularly herds, ruminants inadvertently signal their presence to hard ticks . Here, we report that all adult hard tick species we tested are attracted to cud and demonstrate that these acarines possess olfactory receptor cells for the carboxylic acid, phenol and indole end-products of the rumen bioreactor . Compounds from each of these classes of volatiles attract ticks on their own, and mixtures of these volatiles based on rumen composition also attract . Appetence for rumen metabolites represents a fundamental resource-tracking adaptation by hard ticks for large roaming mammals. Vaccine, 2004 Dec 9, 23(4), 518 - 24 In vitro growth and stability of recombinant rabies viruses designed for vaccination of wildlife; Dietzschold ML et al.; Three live rabies virus (RV) recombinant vaccine candidates, SPBNGA, SPBNGA-Cyto c (+), and SPBNGA-GA, were examined for their production levels and stability . Maximum production levels up to 10(10) infectious particles/mL were achieved using bioreactor technology . All virus lots exhibited thermostability profiles typical for RV vaccines and were non-pathogenic for intracranially inoculated immunocompetent mice . Moreover, sequence analysis indicated high genetic stability in all three RVs during 10 consecutive passages in newborn mice . This analysis revealed no change in the extra RV G gene in the SPBNGA-GA vaccine or in the cytochrome c gene in the SPBNGA-Cyto c (+) vaccine . Moreover, no changes were detected in the G gene codon for Glu333, which renders the virus non-pathogenic . However, after the fifth passage, a mutation resulting in an Asn194 --> Lys194 exchange emerged in the G genes of all three RVs . This mutation was associated with a modest increase in pathogenicity in SPBNGA and SPBNGA-Cyto c (+), but not in SPBNGA-GA, which contained the mutation in only one of its two G genes and which remained non-pathogenic . These results demonstrate the feasibility of producing RV vaccines that remain highly stable even after multiple passages. Vaccine, 2004 Dec 9, 23(4), 511 - 7 Vero-cell rabies vaccine produced using serum-free medium; Frazatti-Gallina NM et al.; A new rabies vaccine was developed from Vero cells adhered to microcarriers, cultivated in a bioreactor in serum-free medium and infected with the PV/VERO-Paris rabies virus strain . The viral suspensions were concentrated by tangential filtration, purified by chromatography and inactivated with beta-propiolactone . In immunogenicity studies performed in mice immunized with three doses of the new vaccine (seven batches) and the commercial Verorab and HDCV, mean titers of neutralizing antibodies of 10.3-34.6, 6.54 and 9.36 IU/ml were found, respectively . The vaccine presented stability during 14 months at 2-8 degrees C, 30 days at 37 degrees C and 8 h at 45 degrees C . The use of serum-free medium facilitated the downstream process leading to residual cellular DNA values <22.8 pg per dose of vaccine in all produced batches . The effective immunogenicity induced in mice by this vaccine, the degree of purity of the product, the high antigen yield and the reduction of the cost of the product due to the virus production and purification processes, makes this technology very important for countries where rabies presents a great public health problem. Folia Microbiol (Praha), 2004, 49(4), 418 - 22 Conidia production of Beauveria sp . by solid-state fermentation for biocontrol of Ilex paraguariensis caterpillars; Dalla Santa HS et al.; Conidia production of Beauveria sp . strain LAG by solid-state fermentation (SSF) using blends of agro-industrial residues (residual potatoes and sugar-cane bagasse) was optimized with respect to cultivation conditions and the composition of substrate mixture in Erlenmeyer flasks and column-type bioreactor . With a blend of 60 % residual potatoes and 40 % sugar-cane bagasse the optimum conditions achieved were: incubation temperature 26 degrees C, initial substrate pH 6, inoculum concentration 10(7) conidia per g substrate; optimal initial moisture of the substrate was 70 % for Erlenmeyer flasks, in column-type bioreactor (with forced aeration) the optimal initial moisture of the substrate was 65 % with airflow of 60 mL/min . The highest production (1.07 x 10(10) conidia per g dry substrate) was achieved after a 10-d fermentation . The conidia were used in laboratory assays against Thelosia camina and Hylesia sp., caterpillars that are serious pests of mate plants . The mortality of T . camina was >90 % 10 d after spraying caterpillars with 1 mL conidia suspension at a concentration 10(5)-10(8)/mL . For Hylesia sp., the mortality was 70 %, 7 d after immersion in the conidia suspension containing 108 conidia per mL . Therefore, the Beauveria sp . LAG can be considered to be an important biocontrol instrument in the prospect of the Integrated Pest Management for mate plants. Nat Biotechnol, 2004 Nov, 22(11), 1393 - 8 Production of recombinant protein therapeutics in cultivated mammalian cells; Wurm FM; Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications because of their capacity for proper protein folding, assembly and post-translational modification . Thus, the quality and efficacy of a protein can be superior when expressed in mammalian cells versus other hosts such as bacteria, plants and yeast . Recently, the productivity of mammalian cells cultivated in bioreactors has reached the gram per liter range in a number of cases, a more than 100-fold yield improvement over titers seen for similar processes in the mid-1980s . This increase in volumetric productivity has resulted mainly from improvements in media composition and process control . Opportunities still exist for improving mammalian cell systems through further advancements in production systems as well as through vector and host cell engineering. Brain Res Dev Brain Res, 2004 Nov 25, 153(2), 163 - 73 Neural stem cell differentiation in a cell-collagen-bioreactor culture system; Lin HJ et al.; Neural stem cells and neural progenitors (NSCs/NPs) are capable of self-renewal and can give rise to both neurons and glia . Such cells have been isolated from the embryonic brain and immobilized in three dimensional collagen gels . The collagen-entrapped NSCs/NPs recapitulate CNS stem cell development and form functional synapses and neuronal circuits . However, the cell-collagen constructs from static conditions contain hypoxic, necrotic cores and the cells are short-lived . In the present study, NSCs/NPs isolated from embryonic day 13 rat cortical neuroepithelium are immobilized in type I collagen gels and cultured in NASA-designed rotating wall vessel (RWV) bioreactors for up to 9 weeks . Initially, during the first 2 weeks of culture, a lag phase of cellular growth and differentiation is observed in the RWV bioreactors . Accelerated growth and differentiation, with the cells beginning to form large aggregates (approximately 1 mm in diameter) without death cores, begins during the third week . The collagen-entrapped NSCs/NPs cultured in RWV show active neuronal generation followed by astrocyte production . After 6 weeks in rotary culture, the cell-collagen constructs contain over 10 fold greater nestin+ and GFAP+ cells and two-fold more TuJ1 gene expression than those found in static cultures . In addition, TuJ1+ neurons in RWV culture give rise to extensive neurite outgrowth and considerably more synapsin I+ pre-synaptic puncta surrounding MAP2+ cell bodies and dendrites . These results strongly suggest that the cell-collagen-bioreactor culture system supports long-term NSC/NP growth and differentiation, and RWV bioreactors can be useful in generating neural tissue like constructs, which may have the potential for cell replacement therapy. Curr Top Dev Biol, 2004, 62, 55 - 85 Blood vessel patterning at the embryonic midline; Hogan KA et al.; The reproducible pattern of blood vessels formed in vertebrate embryos has been described extensively, but only recently have we obtained the genetic and molecular tools to address the mechanisms underlying these processes . This review describes our current knowledge regarding vascular patterning around the vertebrate midline and presents data derived from frogs, zebrafish, avians, and mice . The embryonic structures implicated in midline vascular patterning, the hypochord, endoderm, notochord, and neural tube, are discussed . Moreover, several molecular signaling pathways implicated in vascular patterning, VEGF, Tie/tek, Notch, Eph/ephrin, and Semaphorin, are described . Data showing that VEGF is critical to patterning the dorsal aorta in frogs and zebrafish, and to patterning the vascular plexus that forms around the neural tube in amniotes, is presented . A more complete knowledge of vascular patterning is likely to come from the next generation of experiments using ever more sophisticated tools, and these results promise to directly impact on clinically important issues such as forming new vessels in the human body and/or in bioreactors. J Biotechnol, 2004 Nov 9, 114(3), 315 - 26 CellMAC: a novel technology for encapsulation of mammalian cells in cellulose sulfate/pDADMAC capsules assembled on a transient alginate/Ca2+ scaffold; Weber W et al.; Microencapsulation of desired mammalian cell phenotypes in biocompatible polymer matrices represents a powerful technology for cell-based therapies and biopharmaceutical manufacturing of protein therapeutics . We have pioneered a novel jet break-up-compatible process for encapsulation of mammalian cells in cellulose sulfate (CS)/poly-diallyl-dimethyl-ammoniumchloride (pDADMAC) (CellMAC) capsules . CS and pDADMAC polymerize on a transient ad hoc co-assembled Ca2+/alginate scaffold and form homogenous capsules following dissolution of the alginate core by Ca2+ chelating agents . CellMAC capsules exhibited excellent mechanical properties and showed a molecular weight cut-off between 43 and 77kDa . Chinese hamster ovary cells engineered for constitutive production of the glycohormone erythropoietin reached high viable cell densities when grown inside CellMAC capsules, while specific erythropoietin (EPO) productivities matched those of conventional non-encapsulated control cultures . CellMAC-encapsulated EPO-production cell lines induced increased EPO serum levels when implanted intraperitoneally into mice and provided robust glycoprotein production during standard stirred-tank bioreactor operation . We expect the CellMAC technology to foster advances in therapeutic encapsulation of engineered cell lines as well as manufacturing of protein pharmaceuticals. J Biomech, 2005 Jan, 38(1), 185 - 90 A CFD level-set method for soft tissue growth: theory and fundamental equations; Lappa M; A level-set method, specifically conceived for the case of soft organic tissue growth from feeding solutions, is introduced and described in detail . The model can handle the morphological evolution of the organic specimen under the influence of external convection (fluid-dynamics of the bioreactor) . The analogies and differences between this technique and a previous volume of fraction method are discussed pointing out advantages and limitations of both formulations. Biotechnol Bioeng, 2004 Dec 20, 88(6), 767 - 77 Increased rate of chondrocyte aggregation in a wavy-walled bioreactor; Bueno EM et al.; A novel wavy-walled bioreactor designed to enhance mixing at controlled shear stress levels was used to culture chondrocytes in suspension . Chondrocyte aggregation in suspensions mixed at 30, 50, and 80 rpm was characterized in the wavy-walled bioreactor and compared with that in conventional smooth-walled and baffled-walled spinner flask bioreactors . Aggregation was characterized in terms of the percentage of cells that aggregated over time, and aggregate size changes over time . The kinetics of chondrocyte aggregation observed in the bioreactors was composed of two phases: early aggregation between 0 and 2 h of culture, and late aggregation between 3 and 24 h of culture . At 50 rpm, the kinetics of early aggregation in the wavy-walled bioreactor was approximately 25% and 65% faster, respectively, than those in the smooth-walled and baffled-walled spinner flask bioreactors . During the late aggregation phase, the kinetics of aggregation in the wavy-walled bioreactor were approximately 45% and 65% faster, respectively, than in the smooth-walled and baffled-walled spinner flasks . The observed improved kinetics of chondrocyte aggregation was obtained at no cost to the cell survival rate . Results of computerized image analysis suggest that chondrocyte aggregation occurred initially by the formation of new aggregates via cell-cell interactions and later by the joining of small aggregates into larger cell clumps . Aggregates appeared to grow for only a couple of hours in culture before reaching a steady size, possibly determined by limitations imposed by the hydrodynamic environment . These results suggest that the novel geometry of the wavy-walled bioreactor generates a hydrodynamic environment distinct from those traditionally used to culture engineered cartilage . Such differences may be useful in studies aimed at distinguishing the effects of the hydrodynamic environment on tissue-engineered cartilage . Characterizing the wavy-walled bioreactor's hydrodynamic environment and its effects on cartilage cell/tissue culture can help establish direct relationships between hydrodynamic forces and engineered tissue properties. Biotechnol Appl Biochem . 2004 Oct 26; {Epub ahead of print} Continuous production of monoclonal antibody in a packed-bed bioreactor; Golmakany N et al.; In this study, growth and monoclonal antibody production of a mouse-mouse hybridoma cell producing anti-digoxin monoclonal antibody was evaluated . The hybridoma cells entrapped within the support matrix named Fibracel (R) were cultured in batch and continuous mode following special protocols . Cell culture studies were performed in a 1 litre spinner basket containing 3 g.l -1 of support matrix . Batch culture was operated with the cell density of 42 x 10 6 cells . During the seven days of culture, the medium was sampled daily in order to assess glucose and monoclonal antibody concentrations and the lactate dehydrogenase (LDH) released into the culture medium . After 72 h of culture period, the cell density and monoclonal antibody concentration were found to be 10.4 x 10 7 cells per 3 g of NWPF discs and 250 mug/ml, respectively . This yield gradually decreased to 0.55 x 10 6 cells per 3 g of packaging material and 60 mug/ml respectively at the end of the batch culture . In the continuous culture studies, initially the batch culture was operated for 64.5 h and then continuous flow was started at the dilution rates of 0.15, 0.2, 0.25 and 0.3 d -1 and finally stabilized at 0.25 d -1 within 288 h (12 days) . The monoclonal antibody concentration at steady state was found to be 116-120 mug/ml.d and the yield of operation was 62.5 mg/ml.d, which was 3.5 times higher than that of batch culture . In conclusion, a packed-bed bioreactor with the support matrix of Fibracel (R), operated in continuous feeding, is more efficient for large-scale monoclonal antibody production than a batch culture . On the other hand, by using this system better supplying of nutrients and removal of inhibitory metabolites and proteolytic enzymes were obtained. Apoptosis, 2004 Nov, 9(6), 843 - 52 Suppression of apoptosis in perfusion culture of Myeloma NS0 cells enhances cell growth but reduces antibody productivity; Tey BT et al.; A spin filter perfusion systems was used to achieve a high cell density culture for two NS0 cell lines in 2 litres bioreactors . One cell line is transfected with the bcl-2 gene (NS0 Bcl-2) encodes the 'anti-apoptotic' human Bcl-2 protein and the other cell line (NS0 Control) with a blank vector . The runs started as batch cultures for two days and were perfused with fresh medium at 0.5 volumes per day (day(-1)) for 4 days, increasing gradually to 2 day(-1) at day 7 . The increase of the viable cell density of Bcl-2 cell line was far greater than the control cell line, although they were perfused with the same amount of medium . At the end of the period of each perfusion rate, the viable cell densities of Bcl-2 culture were 30%, 120%, 160% and 220% higher than its control cell line corresponding values . Overall, there was a roughly 9 fold increase in viable cell density from the inoculum for the control culture, but almost a 30 fold increase for the Bcl-2 culture . The mode of cell death in the control culture was initially predominantly by necrosis (viability higher than 80%), but apoptotic cell death became more significant after day 8 of the culture . Cell death in the Bcl-2 culture was almost entirely by necrosis, although it remained at a very low level (less than 5%) to the termination time . The cell cycle distributions for both cell lines were very much similar indicating they have a similar doubling time and G1 to S progression rate . Interestingly, the Bcl-2 cultures exhibited reduced antibody specific production rate with increasing viable cell number and time . The volumetric production rate was, however, similar in both cultures . Bcl-2 as an anti-death protein allowed cells to survive and thus divide to higher cell densities without the need for additional nutrients . Most of the cellular energy in a producer cell line is used for biomass production rather than for antibody production, as was the case with the control cell line. Nat Biotechnol, 2004 Nov, 22(11), 1440 - 4 Epub 2004 Oct 17. Gas-inducible transgene expression in mammalian cells and mice; Weber W et al.; We describe the design and detailed characterization of a gas-inducible transgene control system functional in different mammalian cells, mice and prototype biopharmaceutical manufacturing . The acetaldehyde-inducible AlcR-P(alcA) transactivator-promoter interaction of the Aspergillus nidulans ethanol-catabolizing regulon was engineered for gas-adjustable transgene expression in mammalian cells . Fungal AlcR retained its transactivation characteristics in a variety of mammalian cell lines and reversibly adjusted transgene transcription from chimeric mammalian promoters (P(AIR)) containing P(alcA)-derived operators in a gaseous acetaldehyde-dependent manner . Mice implanted with microencapsulated cells engineered for acetaldehyde-inducible regulation (AIR) of the human glycoprotein secreted placental alkaline phosphatase showed adjustable serum phosphatase levels after exposure to different gaseous acetaldehyde concentrations . AIR-controlled interferon-beta production in transgenic CHO-K1-derived serum-free suspension cultures could be modulated by fine-tuning inflow and outflow of acetaldehyde-containing gas during standard bioreactor operation . AIR technology could serve as a tool for therapeutic transgene dosing as well as biopharmaceutical manufacturing. Zhonghua Yi Xue Za Zhi, 2004 Sep 2, 84(17), 1460 - 4 {Study on the interface of human hepatocyte/micropore polypropylene ultrafiltration membrane}; Peng CH et al.; OBJECTIVE: To found a new interface of human hepatocyte/micropore polypropylene ultrafiltration membrane (MPP) with good cytocompatibility so as to construct bioartificial bioreactor with polypropylene hollow fibers in future . METHODS: MPP ultrafiltration membrane underwent chemical grafting modification through ultraviolet irradiation and Fe(2+) reduction . The contact angles of MPP and the modified MPP membranes were measured . Human hepatic cells L-02 were cultured . MPP and modified MPP membranes were spread on the wells of culture plate and human hepatic cells and cytodex 3 were inoculated on them . Different kinds of microscopy were used to observe the morphology of these cells . RESULTS: The water contact angle of MPP and the modified MPP membranes decreased from 78 degrees +/- 5 degrees to 27 degrees +/- 4 degrees (P < 0.05), which indicated that the hydrophilicity of the membrane was improved obviously after the grafting modification . Human hepatocyte L-02 did not adhere to and spread on the modified MPP membrane surface, and only grew on the microcarrier cytodex 3 with higher density and higher proliferation ratio measured by MTT . CONCLUSION: Grafting modification of acrylamide on MPP membrane is a good method to improve the human hepatocyte cytocompatibility with MPP ultrafiltration membrane. Zhonghua Wai Ke Za Zhi, 2004 Sep 7, 42(17), 1064 - 8 {Study on the interface of human hepatocyte L-02 polypropylene:simple culture method of human hepatocyte with spheroidal aggregate culture.}; Peng CH et al.; OBJECTIVE: To found new interface of human hepatocyte/poly propylene with good cytocompatibility for made polypropylene hollow fibers bioreactor of bioartificial liver in future . METHODS: Using the macromolecular hydroperoxide groups on the polypropylene membrane surface as initiators, acrylamides were polymerized on the polypropylene membranes, under induction by both UV irradiation and Fe2+ reduction . Growth characteristics of human hepatocyte L-02 were detected when it was cultured on polystyrene, polypropylene and modified polypropylene membrane surface . RESULTS: Water contact angle measurement of the polypropylene and the modified polypropylene membranes decreased from (72 +/- 5) degrees to (30 +/- 4) degrees , which indicated that the hydrophilicity of the membrane was improved obviously after the grafting modification . Human hepatocyte L-02 could not adhere and spread on modified polypropylene membrane surface, and grown in spheroidal aggregate with higher density and higher proliferation ratio measured by MTT method . CONCLUSIONS: Acrylamide polymerized on the polypropylene membranes is a good method which not only improved human hepatocytes cytocompatibility but also found a new simple culture method with spheroidal aggregate culture of human hepatocyte. Water Sci Technol, 2004, 50(5), 209 - 18 Modelling the degradation of low concentration pollutants in membrane bioreactors; Peev M et al.; This paper deals with the development of a description of membrane bioreactor systems, focussing on wastewater treatment and removal of specific low concentration persistent organic pollutants (e.g . persistent polar pollutants--P3 compounds) . In the present work we offer a basic model, adapted to Membrane Bioreactor (MBR) conditions, which reflects the biodegradation of recalcitrant (organic) substances . We put a particular focus on organic aromatic pollutants and demonstrate a model parameter estimation approach, with experiments carried out for linear alkylbenzene sulphonates (LAS). Water Sci Technol, 2004, 50(5), 1 - 8 Removal of endocrine disrupting compounds with membrane processes in wastewater treatment and reuse; Wintgens T et al.; Endocrine disrupting compounds can affect the hormone system in organisms and are the subject of environmental and human health concerns . The effluents of wastewater treatment plants contribute to the emission of estrogenically active substances into the environment . Membrane technology, which is an advanced wastewater treatment option, is the subject of this research . The removal techniques under investigation are membrane bioreactors, reverse osmosis, and nanofiltration . Eleven different nanofiltration membranes were tested in the laboratory set-up . The observed retention of NP and BPA ranged between 70% and 100% . The contact angle is an indicator for the hydrophobicity of a membrane, whose influence on the permeability and retention of NP was evident . Regarding the retention of BPA no dependency on the contact angle was observed . Results of the investigation of a full-scale landfill leachate treatment plant indicate a bisphenol A (BPA) removal of more than 98% with membrane bioreactors and reverse osmosis . The mass balance indicates that biological degradation is the most important removal process in the membrane bioreactor configuration. Macromol Biosci, 2004 Oct 20, 4(10), 950 - 6 Gluconic acid production in bioreactor with immobilized glucose oxidase plus catalase on polymer membrane adjacent to anion-exchange membrane; Godjevargova T et al.; Gluconic acid was obtained in the permeate side of the bioreactor with glucose oxidase (GOD) immobilized onto anion-exchange membrane (AEM) of low-density polyethylene grafted with 4-vinylpiridine . The electric resistance of the anion-exchange membranes was increased after the enzyme immobilization on the membrane . The gluconic acid productions were relatively low with the GOD immobilized by any method on the AEM . To increase the enzyme reaction efficiency, GOD was immobilized on membrane of AN copolymer (PAN) adjacent to an anion-exchange membrane in bioreactor . Uses of anion-exchange membrane led to selective removal of the gluconic acid from the glucose solution and reduce the gluconic acid inhibition . The amount of gluconic acid obtained in the permeate side of the bioreactor with the GOD immobilized on the PAN membrane adjacent to the AEM under electrodialysis was about 30 times higher than that obtained with enzyme directly bound to the AEM . The optimal substrate concentration in the feed side was found to be about 1 g/l . Further experiments were carried out with the co-immobilized GOD plus Catalase (CAT) on the PAN membrane adjacent to the AEM to improve the efficiency of the immobilize system . The yield of this process was at least 95% . The storage stability of the co-immobilized GOD and CAT was studied (lost 20% of initial activity for 90 d) . The results obtained clearly showed the higher potential of the dual membrane bioreactor with GOD plus CAT bound to ultrafiltration polymer membrane adjacent to the AEM . Storage stability of GOD activity in GOD plus CAT immobilized on PAN//AEM membranes and on AEM. Biosens Bioelectron, 2004 Oct 15, 20(3), 414 - 23 Doppler optical coherence tomography for measuring flow in engineered tissue; Mason C et al.; The engineering of human tissue represents a major paradigm shift in clinical medicine . Early embodiments of tissue engineering are currently being taken forward to the clinic by production methods that are essentially extensions of laboratory manual procedures . However, to achieve the status of routine large-scale clinical practice, automation and scale-out processes are required . This in turn will require the development of reliable on-line monitoring and control systems . This paper examines one demand of crucial importance, namely the real time in vitro monitoring of the flow characteristics through growing tissue since this has a complex interrelationship . Doppler optical coherence tomography (DOCT) is a recently developed imaging technique for studying the rheological properties of tissues in vivo . Capable of non-invasive imaging in real time with high resolution, it is potentially ideal for the continuous monitoring of engineered tissues in vitro . As a base line, the current status of DOCT in vivo is therefore reviewed . This paper also reports the first preliminary use of DOCT in tissue engineering . The application described involves the imaging of a fully developed laminar flow through a combined tissue fabrication/bioreactor with a tissue-engineered construct (substitute blood vessel) in situ. Biotechnol Bioeng, 2004 Nov 5, 88(3), 332 - 47 Cell cycle kinetics of expanding populations of neural stem and progenitor cells in vitro; Alam S et al.; Neural stem cells (NSCs) are undifferentiated, primitive cells with important potential applications including the replacement of neural tissue lost due to neurodegenerative diseases, including Parkinson's disease, as well as brain and spinal cord injuries, including stroke . We have developed methods to rapidly expand populations of mammalian stem and progenitor cells in neurosphere cultures . In the present study, flow cytometry was used in order to understand cell cycle activation and proliferation of neural stem and progenitor cells in suspension bioreactors . First, a protocol was developed to analyze the cell cycle kinetics of NSCs . As expected, neurosphere cells were found to cycle slowly, with a very small proportion of the cell population undergoing mitosis at any time . Large fractions (65-70%) of the cells were detected in G1, even in rapidly proliferating cultures, and significant fractions (20%) of the cells were in G0 . Second, it was observed that different culturing methods influence both the proportion of neurosphere cells in each phase of the cell cycle and the fraction of actively proliferating cells . The results show that suspension culture does not significantly alter the cell cycle progression of neurosphere cells, while long-term culture (>60 days) results in significant changes in cell cycle kinetics . This suggests that when developing a process to produce neural stem cells for clinical applications, it is imperative to track the cell cycle kinetics, and that a short-term suspension bioreactor process can be used to successfully expand neurosphere cells . Biotechnol Bioeng, 2004 Nov 5, 88(3), 313 - 20 Three-dimensional porous alginate scaffolds provide a conducive environment for generation of well-vascularized embryoid bodies from human embryonic stem cells; Gerecht-Nir S et al.; Differentiation of human embryonic stem cells (hESCs) can be instigated through the formation of embryo-like aggregates in suspension, termed human embryoid bodies (hEBs) . Controlling cell aggregation and agglomeration during hEBs formation has a profound effect on the extent of cell proliferation and differentiation . In a previous work, we showed that control over hEBs formation and differentiation can be achieved via cultivation of hESC suspensions in a rotating bioreactor system . We now report that hEBs can be generated directly from hESC suspensions within three-dimensional (3D) porous alginate scaffolds . The confining environments of the alginate scaffold pores enabled efficient formation of hEBs with a relatively high degree of cell proliferation and differentiation; encouraged round, small-sized hEBs; and induced vasculogenesis in the forming hEBs to a greater extent than in static or rotating cultures . We therefore conclude that differentiation of hEBs can be induced and directed by physical constraints in addition to chemical cues . Biomaterials, 2005 Apr, 26(12), 1405 - 11 Mechano-active tissue engineering of vascular smooth muscle using pulsatile perfusion bioreactors and elastic PLCL scaffolds; Jeong SI et al.; Blood vessels are subjected in vivo to mechanical forces in a form of radial distention, encompassing cyclic mechanical strain due to the pulsatile nature of blood flow . Vascular smooth muscle (VSM) tissues engineered in vitro with a conventional tissue engineering technique may not be functional, because vascular smooth muscle cells (VSMCs) cultured in vitro typically revert from a contractile phenotype to a synthetic phenotype . In this study, we hypothesized that pulsatile strain and shear stress stimulate VSM tissue development and induce VSMCs to retain the differentiated phenotype in VSM engineering in vitro . To test the hypothesis, rabbit aortic smooth muscle cells (SMCs) were seeded onto rubber-like elastic, three-dimensional PLCL {poly(lactide-co-caprolactone), 50:50} scaffolds and subjected to pulsatile strain and shear stress by culturing them in pulsatile perfusion bioreactors for up to 8 weeks . As control experiments, VSMCs were cultured on PLCL scaffolds statically . The pulsatile strain and shear stress enhanced the VSMCs proliferation and collagen production . In addition, a significant cell alignment in a direction radial to the distending direction was observed in VSM tissues exposed to radial distention, which is similar to that of native VSM tissues in vivo, whereas VSMs in VSM tissues engineered in the static condition randomly aligned . Importantly, the expression of SM alpha-actin, a differentiated phenotype of SMCs, was upregulated by 2.5-fold in VSM tissues engineered under the mechano-active condition, compared to VSM tissues engineered in the static condition . This study demonstrates that tissue engineering of VSM tissues in vitro by using pulsatile perfusion bioreactors and elastic PLCL scaffolds leads to the enhancement of tissue development and the retention of differentiated cell phenotype. Biotechnol Bioeng, 2004 Dec 5, 88(5), 593 - 600 Mechanism of extraction of beta-carotene from microalga Dunaliellea salina in two-phase bioreactors; Hejazi MA et al.; We show that it is possible to extract beta-carotene selectively from Dunaliella salina in two-phase bioreactors . The cells continue to produce beta-carotene and the extracted part is substituted by newly produced molecules . This process is called "milking." We performed several experiments to understand the exact mechanism of the extraction process . The results show that direct contact between the cells and the biocompatible organic solvent was not a requirement for the extraction but it accelerated the extraction . Electron microscopy photographs showed an undulated shape of the cell membrane and a space between the cell and the chloroplast membranes in the cells growing in the presence of dodecane (a biocompatible solvent) . Extra-chloroplast beta-carotene globules located in the space between the cell and the chloroplast membranes were observed in these cells as well . It was shown that dodecane was taken up by the cells . The concentration of dodecane in the cells was about 13 pg.cll(-1) . It can be concluded that dodecane uptake by the cells is responsible for the morphological changes in the cells and leads to more activity in the cell membrane . The results suggest two possible modes of extraction . One of the mechanisms is transport of the globules from the chloroplast to the space between the cell and the chloroplast membranes and subsequently from there to the outside by exocytosis . Another possible mode for the extraction could be release of beta-carotene from the globules as a result of alterations in the membrane in response to the uptake of dodecane . beta-Carotene molecules diffuse from the chloroplast to the space between the cell and the chloroplast membranes and from there to the medium either by diffusion or by exocytosis after accumulation in the vesicles. Cell Transplant, 2004, 13(4), 393 - 403 Functional activity of human hepatoma cells transfected with adenovirus-mediated hepatocyte nuclear factor (HNF)-4 gene; Naiki T et al.; Fulminant hepatic failure (FHF) is still associated with high mortality despite recent advances in medical management . There is need of an effective and safe bioartificial liver (BAL) support to help keep patients with FHF alive until an organ becomes available for transplantation or the native liver recovers . The aim of this study was to establish highly functional liver cells by means of transfecting hepatocyte nuclear factor (HNF)-4 gene for the development of BAL . We constructed adenovirus vector carrying rat HNF-4 cDNA, and transfected to hepatoma-derived cell lines, HepG2 and HuH-7, to enforce expression of the exogenous HNF-4 gene . We analyzed expression of HNF-4, HNF-1, and liver-specific genes in cells infected by the adenovirus vector expressing HNF-4 . Adenovirus-mediated HNF-4 gene transfer resulted in increases in expressions of HNF-4, HNF-1, and liver-specific genes such as apolipoproteins, alpha1-antitrypsin (alpha1-AT), phosphoenolpyruvate carboxy-kinase, cytochrome P450 families, and glutamine synthetase in transfected hepatoma cells . Cells overexpressing HNF-4 removed ammonia from medium supplemented with NH4Cl to a greater extent than control cells . These findings demonstrated that transfected cell lines restored differentiated gene expressions and liver-specific function by the overproduction of HNF-4 . HNF-4-overexpressing hepatocyte cell lines are useful for bioreactor of BAL systems. Biotechnol Bioeng, 2004 Nov 20, 88(4), 437 - 50 Process development for a recombinant Chinese hamster ovary (CHO) cell line utilizing a metal induced and amplified metallothionein expression system; Huang EP et al.; The suspension Chinese Hamster Ovary cell line, 13-10-302, utilizing the metallothionein (MT) expression system producing recombinant human growth hormone (hGH) was studied in a serum-free and cadmium-free medium at different fermentation scales and modes of operation . Initial experiments were carried out to optimize the concentration of metal addition to induce the MT promoter . Subsequently, the cultivation of the 13-10-302 cell line was scaled up from spinner flasks into bioreactors, and the cultivation duration was extended with fed-batch and perfusion strategies utilizing 180 microM zinc to induce the promoter controlling expression of recombinant hGH . It was shown that a fed-batch process could increase the maximum cell numbers twofold, from 3.3 to 6.3 x 10(6) cell/mL, over those obtained in normal batch fermentations, and this coupled with extended fermentation times resulted in a fourfold increase in final hGH titer, from 135 +/- 15 to 670 +/- 70 mg/L at a specific productivity q(hGH) value of 12 pg cell(-1)d(-1) . The addition of sodium butyrate increased the specific productivity of hGH in cells to a value of approximately 48 pg cell(-1)d(-1), resulting in a final hGH titer of over a gram per liter during fed-batch runs . A BioSep acoustic cell recycler was used to retain the cells in the bioreactor during perfusion operation . It was necessary to maintain the specific feeding rates (SFR) above a value of 0.2 vvd/(10(6) cell/mL) to maintain the viability and productivity of the 13-10-302 cells; under these conditions the viable cell number increased to over 10(7) cell/mL and resulted in a volumetric productivity of over 120 mg(hGH) L(-1)d(-1) . Process development described in this work demonstrates cultivation at various scales and sustained high levels of productivity under cadmium free condition in a CHO cell line utilizing an inducible metallothionein expression system . (c) 2004 Wiley Periodicals, Inc Biotechnol Bioeng, 2004 Dec 5, 88(5), 547 - 57 Effect of feed and bleed rate on hybridoma cells in an acoustic perfusion bioreactor: part I . Cell density, viability, and cell-cycle distribution; Dalm MC et al.; For the development of optimal perfusion processes the effect of the feed and bleed rate on cell growth in a perfusion bioreactor was studied . The viable-cell density, viability, growth, death, and lysis rate and cell-cycle distribution of a hybridoma cell line producing an IgG1 were studied over a range of specific feed and bleed rates . It was found that the feed and bleed rates applied in the different cultures could be divided into two regions based on the viable-cell density and cell-cycle distribution . The cultures in the first region, low feed rates (0.5 and 1.0 d(-1)) combined with low bleed rates (0.05 and 0.10 d(-1)), were nutrient-limited, as an increase in the feed rate resulted in an increase in the viable-cell density . The cultures in the second region, high feed and bleed rates, were nonnutrient-limited . In this region the viable-cell density decreased more or less linearly with an increase in the bleed rate and was independent of the feed rate . This suggests that the cells were limited by a cell-related factor . Comparison of Trypan-blue dye-exclusion measurements and lactate-dehydrogenase activity measurements revealed that cell lysis was not negligible in this bioreactor set-up . Therefore, lactate-dehydrogenase activity measurements were essential to measure the death rate accurately . The specific growth rate was nearly constant for all tested conditions . The viability increased with an increase of the bleed rate and was independent of the feed rate . Furthermore, the specific productivity of monoclonal antibody was constant under all tested conditions . For the optimal design of a perfusion process it should first be established whether viability is an important parameter . If not, a bleed rate as low as possible should be chosen . If low viabilities are to be avoided, the bleed rate chosen should be higher, with the value depending on the desired viability . Next, the feed rate should be set at such a rate that the cells are just in the nonnutrient-limited region. Kidney Int, 2004 Oct, 66(4), 1578 - 88 Initial clinical results of the bioartificial kidney containing human cells in ICU patients with acute renal failure; Humes HD et al.; BACKGROUND: Acute renal failure (ARF) in intensive care unit patients continues to have mortality rates exceeding 70%, despite hemodialysis or continuous renal replacement therapy (CRRT) . The delivery of cellular metabolic function to CRRT may provide more complete renal replacement therapy, thereby changing the natural history of this disease process . An FDA-approved Phase I/II clinical trial on 10 patients has been completed, and demonstrated that this experimental treatment can be delivered safely for up to 24 hours . METHODS: The bioartificial kidney is a synthetic hemofilter connected in series with a bioreactor cartridge containing approximately 10(9) human proximal tubule cells, as a renal tubule assist device (RAD), within an extracorporeal perfusion circuit utilizing standard hemofiltration pump systems . All 10 patients had ARF and multiorgan failure (MOF), with predicted hospital mortality rates averaging above 85% . RESULTS: Data indicate that the RAD maintains viability, durability, and functionality in this ex vivo clinical setting . The device also demonstrated differentiated metabolic and endocrinologic activity, with glutathione degradation and endocrinologic conversion of 25-OH-D(3) to 1,25-(OH)(2)-D(3) . All but one treated patient with more than a 3-day follow-up in the intensive care unit showed improvement as assessed by acute physiologic scores 1 to 7 days following therapy . Six of the 10 treated patients survived past 30 days . One patient expired within 12 hours after RAD treatment due to his family's request to withdraw ventilatory life support . Three other patients died due to complications from acute or chronic comorbidities unrelated to ARF or RAD therapy . Plasma cytokine levels suggest that RAD therapy produced dynamic and individualized responses in patients . For the subset of patients who had excessive proinflammatory levels, RAD treatment resulted in significant declines in granulocyte colony stimulating factor (G-CSF), interleukin (IL)-6, IL-10, and IL-6/IL-10 ratios . CONCLUSION: The addition of human renal tubule cell therapy to CRRT has been accomplished and demonstrates metabolic activity with systemic effects in patients with ARF and MOF . These initial clinical results are encouraging, so that a randomized, controlled Phase II clinical trial is underway to further assess the clinical safety and efficacy of this new therapeutic approach. Biotechnol Prog, 2004 Sep-Oct, 20(5), 1583 - 7 Chlorpromazine N-demethylation by hydroperoxidase activity of covalent immobilized lipoxygenase; Pinto MC et al.; This work describes the application of the N-demethylase activity of immobilized soybean lipoxygenase to the oxidative degradation of xenobiotics . Previously (1) we have shown that immobilized lipoxygenase produces the oxidative degradation of CPZ in the presence of hydrogen peroxide . As a continuation of this work, here we studied the N-demethylation of CPZ by the hydroperoxidase activity of covalent immobilized soybean lipoxygenase . The obtained results clearly reveal that the immobilized system produces the N-demethylation of CPZ in the presence of hydrogen peroxide, maintaining a high level of activity in comparison with free enzyme . Additionally, the immobilized lipoxygenase shows stability higher than that of free enzyme, making feasible its use in a bioreactor operating in continuous or discontinuous mode . The results obtained in this work, together with those obtained previously by us for the oxidation of CPZ, suggest that hydroperoxidase activity of immobilized lipoxygenase may constitute a valuable tool for oxidative xenobiotics degradation or for application to synthetic processes in which a N-demethylation reaction is involved. Biotechnol Prog, 2004 Sep-Oct, 20(5), 1454 - 9 Simulated weightlessness in the design and exploitation of a NMR-compatible bioreactor; Bradamante S et al.; Mammalian cells cultured in simulated weightlessness take advantage of a favorable environment, experiencing low shear stress and reduced turbulence . NMR spectroscopy allows the on-line noninvasive monitoring of cell growth and metabolism . With this in mind, we developed a novel bioreactor that fits into a NMR instrument and in which the simulated weightlessness conditions are obtained by a suitable medium and a flow-lift suspension . In detail, the gravitational vector acting on cells is counterbalanced by the hydrodynamic thrusts created by a bottom-up spiral flow of a fluid having increased density . We validate its efficiency (a) by calculating the main physical parameters as relative velocity, shear stress, and oxygen transport, and (b) by comparing the experimental results of growing a cell culture in the proposed bioreactor with those obtained using an established simulated weightlessness system (rotating wall vessel, NASA) . As a test study we focused on the proliferation of human umbilical vein endothelial cells (HUVEC) in terms of cell viability and organization of their cytoskeleton. Biotechnol Prog, 2004 Sep-Oct, 20(5), 1449 - 53 Kinetics of Gibberella fujikuroi growth and gibberellic acid production by solid-state fermentation in a packed-bed column bioreactor; Machado CM et al.; In this work the growth of Gibberella fujikuroi and gibberellic acid (GA3) production were studied using coffee husk and cassava bagasse as substrates in a packed-bed column bioreactor connected to a gas chromatograph for exit gas analysis . With the respirometric data, a logarithmic correlation between accumulated CO2 and biomass production was determined, and the kinetics of the fungal growth was compared for estimated and experimental data . The solid medium consisted of coffee husk (pretreated with alkali solution), mixed with cassava bagasse (7:3 dry weight basis), with a substrate initial pH of 5.2 and moisture of 77% . Cultivation was carried out in glass columns, which were packed with preinoculated substrate and with forced aeration of 0.24 L of air/{h (g of substrate)} for the first 3 days, and 0.72 L of air/{h (g of substrate)} for the remaining period . The maximum specific growth rate (microm) obtained was 0.052 h(-1) (between 24 and 48 h of fermentation) . A production of 0.925 g of GA3/kg of substrate was achieved after 6 days of fermentation. Biotechnol Prog, 2004 Sep-Oct, 20(5), 1408 - 13 Hyperproduction of cordycepin by two-stage dissolved oxygen control in submerged cultivation of medicinal mushroom Cordyceps militaris in bioreactors; Mao XB et al.; Effect of oxygen supply on cordycepin production was investigated in submerged cultivation of Cordyceps militaris, a famous traditional Chinese medicinal mushroom, in a 5-L turbine-agitated bioreactor (TAB) . Initial volumetric oxygen transfer coefficient (kLa) within the range of 11.5-113.8 h(-1) had significant influence on cordycepin production . The highest cordycepin concentration of 167.5 mg/L was obtained at an initial kLa value of 54.5 h(-1), where a moderate dissolved oxygen (DO) pattern was observed throughout cultivation . The possible correlation between cordycepin production and DO level was explored by DO control experiments, and the results showed that DO within the range of 10-80% of air saturation greatly affected the cultivation process . To obtain a high specific cordycepin formation rate (rho) throughout cultivation, a two-stage DO control strategy was developed based on the analysis of the relationship of rho and DO . That is, DO was controlled at 60% from the beginning of cultivation and then shifted to a lower control level of 30% when rho started to decrease . As a result, a high cordycepin production of 201.1 mg/L and a high productivity of 15.5 mg/(L.d) were achieved, which was enhanced by about 15% and 30% compared to the highest titers obtained in conventional DO control experiments, respectively . The proposed DO control strategy was also applied to a recently developed 5-L centrifugal impeller bioreactor (CIB) with cordycepin production and productivity titers of 188.3 mg/L and 14.5 mg/(L.d) . Furthermore, the scale-up of the two-stage DO control process from 5-L CIB to 30-L CIB was successfully demonstrated . The work is useful for the efficient large-scale production of bioactive metabolites by mushroom cultures. Biotechnol Prog, 2004 Sep-Oct, 20(5), 1402 - 7 Production of a secreted glycoprotein from an inducible promoter system in a perfusion bioreactor; Lipscomb ML et al.; The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture . During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture . Induction of the culture at high cell density ensures that the volumetric production will be maximized . In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system . Retention of suspension-adapted CHO cells was achieved by inclined sedimentation . To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction . This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture . The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor . Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated. Biotechnol Prog, 2004 Sep-Oct, 20(5), 1393 - 401 Production of low-lactose milk by means of nonisothermal bioreactors; Grano V et al.; The effect of the immobilization time on the activity of immobilized beta-galactosidase from K . lactis was investigated . Six biocatalytic membranes, different only for the time of the enzyme immobilization, were obtained by using nylon membranes grafted with glycidyl methacrylate (GMA) and activated by hexamethylenediamine (HMDA) and glutaraldehyde (Glu), used as spacer and coupling agent, respectively . Comparison between the isothermal and nonisothermal yield of these biocatalytic membranes was carried out in the process of lactose hydrolysis in milk . All of the results, reported as a function of the immobilization time, have evidenced the influence of our variable parameter on the activity of the catalytic membranes . The membrane giving highest yield under isothermal and nonisothermal conditions was that obtained with 2 h of immobilization time . The industrial application of these membranes has been discussed in terms of percentage reduction of the production times. Adv Biochem Eng Biotechnol, 2004, 91, 171 - 89 Transgenic birds for the production of recombinant proteins; Kamihira M et al.; Transgenic birds were expected to be an excellent transgenic bioreactor for the production of recombinant pharmaceutical proteins . However, the only successful transgenic bioreactors have been based on mammals . We have developed two key techniques for obtaining transgenic birds . For bird embryo culture, we identified that the low rate of hatchability of cultured embryos is caused by limited oxygen and calcium availability . In quail embryo culture using a chicken eggshell as a culture vessel, hatchability increased to 80% by the supplement of calcium lactate in addition to oxygen aeration . A fully artificial vessel for quail embryo culture using a gas-permeable Teflon membrane was also designed . Although the hatchability was lower than that of cultures using a surrogate eggshell, we succeeded in hatching of bird embryos using a fully artificial vessel . For transgene introduction, a replication-defective pantropic retroviral vector based on Moloney murine leukemia virus (MoMLV) pseudotyped with vesicular stomatitis virus G protein (VSV-G) was injected to laid embryos at the blastodermal stage, and the embryos were hatched in vitro to generate G0 birds . The viral vector sequence was detected in the tissues of all G0 birds . The germ-line transmission efficiency was more than 80% . Plural copies of the transgene were inserted into the genome of G1 transgenic progeny. Adv Biochem Eng Biotechnol, 2004, 91, 135 - 69 Development of culture techniques of keratinocytes for skin graft production; Masahiro KO et al.; The in vitro cultures of human tissues have attracted a great deal of medical attention as a promising technique for repairing defective tissues in vivo . In the last decade many companies have been established for supplying the regenerated grafts by means of tissue cultures of skin, cartilage, bone and so on . From the viewpoint of biochemical engineering, however, the culture systems for these tissues are not so sophisticated nor so programmed as the submerged culture systems developed for microorganisms . In manufacturing skin grafts, for instance, the raw materials of cells harvested from patients are heterogeneous, and the products of cultured tissues vary in the required size for individual epithelial sheets . Therefore, a reliable and robust process is desired for the production of cultured tissues with high reproducibility and quality . This review focuses on the strategies for developing the culture processes of keratinocytes targeting the epithelial sheet production, including (i) the introduction of culture techniques for keratinocyte cells and survey of skin graft production as it is, (ii) construction of kinetic model of cell growth, (iii) evaluation of cell properties based on image-analyzing techniques, and (iv) design of bioreactor system. Adv Biochem Eng Biotechnol, 2004, 91, 105 - 34 Large-scale micropropagation system of plant cells; Honda H et al.; Plant micropropagation is an efficient method of propagating disease-free, genetically uniform and massive amounts of plants in vitro . The scale-up of the whole process for plant micropropagation should be established by an economically feasible technology for large-scale production of them in appropriate bioreactors . It is necessary to design suitable bioreactor configuration which can provide adequate mixing and mass transfer while minimizing the intensity of shear stress and hydrodynamic pressure . Automatic selection of embryogenic calli and regenerated plantlets using image analysis system should be associated with the system . The aim of this chapter is to identify the problems related to large-scale plant micropropagation via somatic embryogenesis, and to summarize the micropropagation technology and computer-aided image analysis . Viscous additive supplemented culture, which is including the successful results obtained by us for callus regeneration, is also introduced. Adv Biochem Eng Biotechnol, 2004, 91, 75 - 103 Large-scale production of hairy root; Uozumi N; Many products of interest are synthesized in organized tissues, but not formed in suspension or callus culture . Therefore, most attention has been focused on root cultures . The transgenic plant,"hairy root", has brought us to dramatic improvements in growth rate and high content of desirable products . Since the roots are quite different from callus in morphology, the culture manner should be explored independently . By providing a growth environment, an elite hairy root can be a more attractive plant . Both of strain selection to generate more competent plants in breeding and engineering development are necessary to overcome various limitations . In this chapter the engineering issues involved in using hairy root culture are discussed, as follows . 1 . Measurement of cell concentration on line, and a designing bioreactors for hairy root in liquid culture . 2 . High cell density culture and its kinetic parameters . 3 . Secretion of target products . 4 . The micropropagation of the regenerated hairy root by means of artificial seed system . In some cases where callus and suspension culture show negligible productivity, organ culture will be necessary to achieve good formation . This study on hairy root culture indicates one of the best attempts to the recovery of products from the organ culture in plant biotechnology. Appl Opt, 2004 Sep 1, 43(25), 4862 - 73 Holographic optical coherence imaging of rat osteogenic sarcoma tumor spheroids; Yu P et al.; Holographic optical coherence imaging is a full-frame variant of coherence-domain imaging . An optoelectronic semiconductor holographic film functions as a coherence filter placed before a conventional digital video camera that passes coherent (structure-bearing) light to the camera during holographic readout while preferentially rejecting scattered light . The data are acquired as a succession of en face images at increasing depth inside the sample in a fly-through acquisition . The samples of living tissue were rat osteogenic sarcoma multicellular tumor spheroids that were grown from a single osteoblast cell line in a bioreactor . Tumor spheroids are nearly spherical and have radial symmetry, presenting a simple geometry for analysis . The tumors investigated ranged in diameter from several hundred micrometers to over 1 mm . Holographic features from the tumors were observed in reflection to depths of 500-600 microm with a total tissue path length of approximately 14 mean free paths . The volumetric data from the tumor spheroids reveal heterogeneous structure, presumably caused by necrosis and microcalcifications characteristic of some human avascular tumors. Biotechnol Bioeng, 2004 Oct 20, 88(2), 248 - 60 Trickle-bed root culture bioreactor design and scale-up: growth, fluid-dynamics, and oxygen mass transfer; Ramakrishnan D et al.; Trickle-bed root culture reactors are shown to achieve tissue concentrations as high as 36 g DW/L (752 g FW/L) at a scale of 14 L . Root growth rate in a 1.6-L reactor configuration with improved operational conditions is shown to be indistinguishable from the laboratory-scale benchmark, the shaker flask (mu=0.33 day(-1)) . These results demonstrate that trickle-bed reactor systems can sustain tissue concentrations, growth rates and volumetric biomass productivities substantially higher than other reported bioreactor configurations . Mass transfer and fluid dynamics are characterized in trickle-bed root reactors to identify appropriate operating conditions and scale-up criteria . Root tissue respiration goes through a minimum with increasing liquid flow, which is qualitatively consistent with traditional trickle-bed performance . However, liquid hold-up is much higher than traditional trickle-beds and alternative correlations based on liquid hold-up per unit tissue mass are required to account for large changes in biomass volume fraction . Bioreactor characterization is sufficient to carry out preliminary design calculations that indicate scale-up feasibility to at least 10,000 liters. Biotechnol Bioeng, 2004 Oct 20, 88(2), 176 - 88 Heterogeneous conditions in dissolved oxygen affect N-glycosylation but not productivity of a monoclonal antibody in hybridoma cultures; Serrato JA et al.; It is known that heterogeneous conditions exist in large-scale animal cell cultures . However, little is known about how heterogeneities affect cells, productivities, and product quality . To study the effect of non-constant dissolved oxygen tension (DOT), hybridomas were subjected to sinusoidal DOT oscillations in a one-compartment scale-down simulator . Oscillations were forced by manipulating the inlet oxygen partial pressure through a feedback control algorithm in a 220-mL bioreactor maintained at a constant agitation . Such temporal DOT oscillations simulate spatial DOT gradients that can occur in large scales . Different oscillation periods, in the range of 800 to 12,800 s (axis of 7% (air saturation) and amplitude of 7%), were tested and compared to constant DOT (10%) control cultures . Oscillating DOT decreased maximum cell concentrations, cell growth rates, and viability indexes . Cultures at oscillating DOT had an increased glycolytic metabolism that was evidenced by a decrease in yield of cells on glucose and an increase in lactate yield . DOT gradients, even several orders of magnitude higher than those expected under practical large-scale conditions, did not significantly affect the maximum concentration of an IgG(1) monoclonal antibody (MAb) . The glycosylation profile of the MAb produced at a constant DOT of 10% was similar to that reported in the literature . However, MAb produced under oscillating culture conditions had a higher amount of triantennary and sialylated glycans, which can interfere with effector functions of the antibody . It was shown that transient excursions of hybridomas to limiting DOT, as occurs in deficiently mixed large-scale bioreactors, is important to culture performance as the oscillation period, and thus the time cells spent at low DOT, affected cell growth, metabolism, and the glycosylation pattern of MAb . Such results underline the importance of monitoring protein characteristics for the development of large-scale processes. Ann Biomed Eng, 2004 Aug, 32(8), 1039 - 49 Design and hydrodynamic evaluation of a novel pulsatile bioreactor for biologically active heart valves; Hildebrand DK et al.; Biologically active heart valves (tissue engineered and recellularized tissue-derived heart valves) have the potential to offer enhanced function when compared to current replacement value therapies since they can possibly remodel, and grow to meet the needs of the patient, and not require chronic medication . However, this technology is still in its infancy and many fundamental questions remain as to how these valves will function in vivo . It has been shown that exposing biologically active tissue constructs to pulsatile pressures and flows during in vitro culture produces enhanced extracellular matrix protein expression and cellularity, although the ideal hydrodynamic conditioning regime is as yet unknown . Moreover, in vitro organ-level studies of living heart valves aimed at studying the remodeling processes require environments that can accurately reproduce in vivo hemodynamics under sterile conditions . To this end, we have developed a system to study the effects of subjecting biologically active heart valves to highly controlled pulsatile pressure and flow waveforms under sterile conditions . The device fits inside a standard incubator and utilizes a computer-controlled closed loop feedback system to provide a high degree of control . The mean pressure, mean flow rate, driving frequency, and shape of the pulsatile pressure waveform can be changed automatically in order to simulate both physiologic and nonphysiologic hemodynamic conditions . Extensive testing and evaluation demonstrated the device's ability to subject a biologically active heart valve to highly controlled pulsatile waveforms that can be modulated during the course of sterile incubation. J Biomed Mater Res, 2004 Nov 15, 71B(2), 313 - 21 Cartilage tissue engineering on the surface of a novel gelatin-calcium-phosphate biphasic scaffold in a double-chamber bioreactor; Chang CH et al.; Tissue engineering is a new approach to articular cartilage repair; however, the integration of the engineered cartilage into the host subchondral bone is a major problem in osteochondral injury . The aim of the present work, therefore, was to make a tissue-engineered osteochondral construct from a novel biphasic scaffold in a newly designed double-chamber bioreactor . This bioreactor was designed to coculture chondrocytes and osteoblasts simultaneously . The aim of this study was to prove that engineered cartilage could be formed with the use of this biphasic scaffold . The scaffold was constructed from gelatin and a calcium-phosphate block made from calcined bovine bone . The cartilage part of the scaffold had a uniform pore size of about 180 microm and approximate porosity of 75%, with the trabecular pattern preserved in the bony part of the scaffold . The biphasic scaffolds were seeded with porcine chondrocytes and cultured in a double-chamber bioreactor for 2 or 4 weeks . The chondrocytes were homogeneously distributed in the gelatin part of the scaffold, and secretion of the extracellular matrix was demonstrated histologically . The chondrocytes retained their phenotype after 4 weeks of culture, as proven immunohistochemically . After 4 weeks of culture, hyaline-like cartilage with lacuna formation could be clearly seen in the gelatin scaffold on the surface of the calcium phosphate . The results show that this biphasic scaffold can support cartilage formation on a calcium-phosphate surface in a double-chamber bioreactor, and it seems reasonable to suggest that there is potential for further application in osteochondral tissue engineering . (c) 2004 Wiley Periodicals, Inc. J Biotechnol, 2004 Sep 30, 113(1-3), 231 - 45 Monitoring and control of Gluconacetobacter xylinus fed-batch cultures using in situ mid-IR spectroscopy; Kornmann H et al.; A partial least-squares calibration model, relating mid-infrared spectral features with fructose, ethanol, acetate, gluconacetan, phosphate and ammonium concentrations has been designed to monitor and control cultivations of Gluconacetobacter xylinus and production of gluconacetan, a food grade exopolysaccharide (EPS) . Only synthetic solutions containing a mixture of the major components of culture media have been used to calibrate the spectrometer . A factorial design has been applied to determine the composition and concentration in the calibration matrix . This approach guarantees a complete and intelligent scan of the calibration space using only 55 standards . This calibration model allowed standard errors of validation (SEV) for fructose, ethanol, acetate, gluconacetan, ammonium and phosphate concentrations of 1.16 g/l, 0.36 g/l, 0.22 g/l, 1.54 g/l, 0.24 g/l and 0.18 g/l, respectively . With G . xylinus, ethanol is directly oxidized to acetate, which is subsequently metabolized to form biomass . However, residual ethanol in the culture medium prevents bacterial growth . On-line spectroscopic data were implemented in a closed-loop control strategy for fed-batch fermentation . Acetate concentration was controlled at a constant value by feeding ethanol into the bioreactor . The designed fed-batch process allowed biomass production on ethanol . This was not possible in a batch process due to ethanol inhibition of bacterial growth . In this way, the productivity of gluconacetan was increased from 1.8 x 10(-3) {C-mol/C-mol substrate/h} in the batch process to 2.9 x 10(-3) {C-mol/C-mol substrate/h} in the fed-batch process described in this study. J Biotechnol, 2004 Sep 30, 113(1-3), 69 - 76 A genome-wide transcription analysis of a fungal riboflavin overproducer; Karos M et al.; The production of many fine chemicals such as vitamins and amino acids is carried out in bioreactors using microorganisms . Usually, these strains are developed from wild-type organisms by classical mutation and selection . After several generations of strain improvement, no further enhancement can be achieved . Therefore, metabolic engineering (ME) is a rational approach to optimise such producer organisms beyond this point, or for starting all over from the beginning . Metabolic Engineering involves detailed analysis of the organism's metabolic and genetic properties, leading to the identification of new target genes . The fungal riboflavin overproducer Ashbya gossypii converts vegetable oil to vitamin B2 in a "one-step reaction" . The productivity and selectivity of this microorganism have been optimised significantly over the years, first following a classical approach and now a rational one . The improvement is based on our understanding of vitamin B2 metabolism . We have been able to selectively enhance the pathways that are necessary for the formation of riboflavin and to inhibit those leading to unwanted side products . New targets for further improvements of this process have been found using a genome-wide transcript expression analysis; namely massive parallel signature sequencing (MPSS) . With this analysis even completely unknown genes can be used for strain improvement. J Chromatogr A, 2004 Aug 6, 1045(1-2), 99 - 109 Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins; Calleri E et al.; The preparation and characterization of a new trypsin-based bioreactor is here described for on-line protein digestion and peptide analysis . Trypsin was immobilized on an epoxy-modified silica monolithic support with a single reaction step and the amount of immobilized enzyme was found to be 66.07 mg (+/-11.75 S.D.)/column (n = 6) . The bioreactor was coupled through a switching valve to an analytical column for the on-line digestion, peptide separation and identification of test proteins by ESI-MS-MS . The influence of various parameters (flow rate, temperature, buffer pH and molarity, etc.) on enzymatic activity was investigated by an experimental design and the mostly significant factor was found to be the flow rate . The efficacy of the reported on-line bioreactor for tryptic mapping is reported for somatostatin and myoglobin, selected as model compounds . Tryptic peptide maps obtained by on-line digestion of myoglobin were compared to those obtained by traditional off-line digestion . Sequence coverage obtained with the on-line protocol (21 peptides, 75.16% coverage of myoglobin sequence) was found to be comparable to the one obtained with the off-line protocol (18 peptides, 76.47% coverage) . Sensitivity for myoglobin digestion and identification was 0.1 mg/ml . The reproducibily of the peptide maps in terms of retention time was from 1.53 to 4.31%, R.S.D. Biomed Microdevices, 2004 Sep, 6(3), 241 - 9 Evaluation of the paratrend multi-analyte sensor for potential utilization in long-duration automated cell culture monitoring; Hwang EY et al.; BACKGROUND: Compact and automated sensors are desired for assessing the health of cell cultures in biotechnology experiments . While several single-analyte sensors exist to measure culture health, a multi-analyte sensor would simplify the cell culture system . One such multi-analyte sensor, the Paratrend 7 manufactured by Diametrics Medical, consists of three optical fibers for measuring pH, dissolved carbon dioxide (pCO(2)), dissolved oxygen (pO(2)), and a thermocouple to measure temperature . The sensor bundle was designed for intra-vascular measurements in clinical settings, and can be used in bioreactors operated both on the ground and in NASA's Space Shuttle and International Space Station (ISS) experiments . METHODS: A Paratrend 7 sensor was placed at the outlet of a bioreactor inoculated with BHK-21 (baby hamster kidney) cells . The pH, pCO(2), pO(2), and temperature data were transferred continuously to an external computer . Cell culture medium, manually extracted from the bioreactor through a sampling port, was also assayed using a bench top blood gas analyzer (BGA) . RESULTS: Two Paratrend 7 sensors were used over a single cell culture experiment (64 days) . When compared to the manually obtained BGA samples, the sensor had good agreement for pH, pCO(2), and pO(2) with bias (and precision) 0.005(0.024), 8.0 mmHg (4.4 mmHg), and 11 mmHg (17 mmHg), respectively for the first two sensors . A third Paratrend sensor (operated for 141 days) had similar agreement (0.02+/-0.15 for pH, -4+/-8 mm Hg for pCO(2), and 24+/-18 mmHg for pO(2)) . CONCLUSION: The resulting biases and precisions are com- parable to Paratrend sensor clinical results . Although the pO(2) differences may be acceptable for clinically relevant measurement ranges, the O(2) sensor in this bundle may not be reliable enough for the ranges of pO(2) in these cell culture studies without periodic calibration. Int J Mol Med, 2004 Oct, 14(4), 663 - 8 Expression of CYP3A4 by an immortalized human hepatocyte line in a three-dimensional culture using a radial-flow bioreactor; Akiyama I et al.; Cytochrome P450 (CYP) 3A is responsible for about 50% of drug metabolizing activity in the liver . The present study was undertaken to establish a CYP3A4-active model for in vitro analysis of human drug metabolism . The cells used were immortalized normal human fetal hepatocytes (OUMS-29) and its HNF4alpha-introduced subline (OUMS-29/H-11) . The cells were cultivated under high-density three-dimensional conditions in a radial-flow bioreactor (RFB) . The number of OUMS-29 cells increased 15-fold over 49 days and their apical surfaces were covered with abundant microvilli, a characteristic of hepatocytes in vivo . The amount of albumin secreted by OUMS-29 cells in the three-dimensional RFB culture was 6-fold higher than those in a monolayer culture . CYP3A4 protein and an intermediate metabolite of testosterone by CYP3A4 were detected in OUMS-29/H11 cells cultivated in RFB >29 days . These results indicate that the RFB culture of OUMS-29/H-11 cells is useful for screening and developing new drugs. J Biomed Mater Res, 2004 Oct 1, 71A(1), 128 - 33 HEK 293 cell suspension culture using fibronectin-adsorbed polymer nanospheres in serum-free medium; Ryu JH et al.; Previously, we reported on suspension culture of anchorage-dependent animal cells using plain polymer nanospheres in serum-containing medium . For commercial cell culture, it is more advantageous to use serum-free medium than serum-containing medium . To culture anchorage-dependent animal cells using polymer nanospheres in serum-free medium, the nanospheres need to be coated with cell adhesion proteins . In this study, we utilized fibronectin-adsorbed polymer nanospheres for suspension culture of anchorage-dependent animal cells in serum-free medium . Fibronectin was adsorbed onto poly(lactic-co-glycolic acid) nanospheres (433 nm in average diameter) by immersing the nanospheres in fetal bovine serum . The nanospheres were used to culture human embryonic kidney (HEK) 293 cells in serum-free medium in stirred suspension bioreactors . Nanospheres attached between HEK 293 cells and promoted cell aggregate formation compared with culture without nanospheres . Most cells in the aggregates were viable over a 10-day culture period . Importantly, the use of poly(lactic-co-glycolic acid) nanospheres promoted the cell growth significantly, compared with culture without nanospheres (3.8- vs 1.8-fold growth) . The nanosphere culture method developed in this study removes the time-consuming and costly process of adaptation of anchorage-dependent animal cells to suspension culture in serum-free medium . This culture method may be useful for the large-scale suspension culture of various types of anchorage-dependent animal cells in serum-free medium. Handchir Mikrochir Plast Chir, 2004 Aug, 36(4), 205 - 11 {In vitro fabrication of tendon substitutes using human mesenchymal stem cells and a collagen type I gel}; Kall S et al.; BACKGROUND AND PURPOSE: Reconstruction of tendon tissue is problematic in many cases . Since direct tendon suture is often impossible, major reconstruction with the use of free tendon transplants or tendon transposition is necessary . Important motor units often have to be sacrificed for reconstructive purposes . In this study we investigated whether long tendon-like substitutes can be fabricated in vitro from human mesenchymal stem cells (MSCs) and a collagen type I gel when cultured under cyclic stretching conditions . MATERIAL AND METHODS: MSCs were obtained from bone marrow aspirates of the iliac crest . Cells were suspended in a collagen type I gel and polymerized in a glass-cylinder with defined size . The fabricated tendon substitutes underwent static stretching for 14 days followed by cyclic stretching for 21 days in a special manufactured bioreactor . Non-stretched substitutes served as a control . RESULTS: Macroscopically the stretched tendon substitutes showed an increased opacity and a smoother surface structure compared to the non-stretched control . The stretched substitutes displayed more spindle-shaped, longitudinal orientated cells, a tendon-like organization of the collagen matrix, and a parallel organization of the collagen fibers when stained with Hematoxylin/Eosin and Elastica . CONCLUSION: Long tendon substitutes could be fabricated from MSCs and a collagen type I gel by cyclic stretching and showed tendon-like parallel collagen fibers and spindle-shaped cells . The use of MSCs in combination with adequate scaffold materials has great therapeutic potential for the development of autologous transplantable tendon substitutes. Vaccine, 2004 Sep 28, 22(29-30), 3858 - 64 Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development; Wu SC et al.; Enterovirus 71 (EV71) is an enterovirus that could lead to severe neurological disorders and fatalities . The inactivated vaccine is an appropriate EV71 vaccine format for meeting current needs . Large-scale preparation of the inactivated EV71vaccine depends on a scalable cell culture system for industrial mass production . In this paper, Vero cells were found to produce higher titers of EV71 than did MRC-5 and WI-38 cells . High-density microcarrier Vero cell cultures were established using 5g/L Cytodex 1 microcarriers and found to promote the release of EV71s from infected Vero cells . For the large-scale production of the inactivated vaccine antigen, the extracellular virus titers produced in the 2L bioreactor were found to be 10 times lower than the spinner flask culture but improved by 30-folds using glucose/glutamine feedings during infection . A serum-free Vero cell microcarrier culture was also established in the bioreactor, yielding a high-titer of 5.8 x 10(7) TCID50/mL for EV71 production . The immunogenicity of the inactivated virions produced in serum-free culture elicited a slightly higher level of neutralizing antibody response in immunized mice . These results constitute valuable information on the development of a large-scale microcarrier cell culture process for producing inactivated EV71 vaccine. Tissue Eng, 2004 Jul-Aug, 10(7-8), 1224 - 33 Novel pulse duplicating bioreactor system for tissue-engineered vascular construct; Narita Y et al.; Cell culture in a biomimetic environment is known to improve the mechanical endurance of tissue-engineered cardiovascular components . Our goal was to generate a bioreactor that can reproduce a wide range of pulsatile flows with a completely physiological pressure profile . The morphology and biochemical properties of tissue-engineered products were also studied to test the usefulness of this novel bioreactor . The combination of an outflow valve, compliance chamber, and resistant clamps together with a balloon pumping system was able to successfully reproduce both physiological systolic and diastolic pressures . The compliance chamber was especially effective in transforming the original peaky pressure waveform into a physiological pressure profile . The tissues, cultured under a physiological pressure waveform with pulsatile flow, presented widely distributed cells in close contact with each other . They also showed significantly higher cell numbers, total protein content, and proteoglycan-glycosaminoglycan content than cultured tissues under a peaky pressure wave or under static conditions . This new bioreactor system is suitable for evaluating a favorable environment for tissue-engineered cardiovascular components. Tissue Eng, 2004 Jul-Aug, 10(7-8), 1113 - 24 Time course of primary liver cell reorganization in three-dimensional high-density bioreactors for extracorporeal liver support: an immunohistochemical and ultrastructural study; Zeilinger K et al.; To enable extracorporeal liver support based on the use of primary liver cells, culture models supporting the maintenance of cell integrity and function in vitro are required . In this study the cell organization and ultrastructure of primary porcine hepatocytes cocultured with nonparenchymal cells in three-dimensional high-density bioreactors were analyzed after 10, 20, and 30 days of culture by immunohistochemistry and transmission electron microscopy . Biochemical data showed that metabolic activity of the cells in the system was relatively stable over at least 20 days . Immunohistochemical studies were performed in comparison with donor organ biopsies . They showed that hepatocytes and nonparenchymal cells reaggregated in bioreactors, forming structures partly resembling natural liver parenchyma . Bile duct-like structures characterized by cytokeratin 7 (CK-7) immunoreactivity (IR) were regularly detected . Nonparenchymal cells (vimentin IR) formed sinusoidal-like str