Bead-to-bead transfer of chinese hamster ovary cells using macroporous microcarriers

Chinese hamster ovary cells (CHO-K1) were cultivated in macroporous gelatin microcarriers (CultiSpher G and CultiSpher S) in spinner flasks and a 5 1 bioreactor. Near-to-confluent cultures were harvested by bead-to-bead transfer where intact microcarriers with cells were transferred from a spinner flask to another spinner flask or to the bioreactor with naked microcarrier beads. Successful bead-to-bead transfer was achieved in various split ratios. The duration of attachment seemed to be important where the direct contact of beads to each other can be achieved by intermittent stirring. Repeated transfers were performed and at least four transfers in spinner flasks were achieved.Two variations of bead-to-bead transfer were performed in the 5 1 bioreactor either by seeding the bioreactor with near-to-confluent beads cultivated in spinner flasks orin situ transfer by adding fresh beads to the bioreactor. As in the spinner case, attachment was achieved by intermittent stirring where donor beads were in close proximity to the acceptor beads. Again successful transfers were obtained as evidenced by the good growth on acceptor beads where cell yields were in the range of 3100–4500 cells/bead.The results suggest that bead-to-bead transfer of CHO-K1 cells can be easily performed and do provide an alternative route to applications where dissolution techniques may not offer an efficient solution.     

Performance of an external loop air-lift bioreactor for the production of monoclonal antibodies by immobilized hybridoma cells

Summary The performance of an external loop air-lift bioreactor was investigated by assessing the inter-relationships between various hydrodynamic properties and mass transfer. The feasibility of using this bioreactor for the production of monoclonal antibodies by mouse hybridoma cells immobilized in calcium alginate gel beads and alginate/poly-l-lysine microcapsules was also examined. When the superficial gas velocity, V _g , in the 300 ml reactor was varied from 2 to 36 cm/min, the average liquid velocity increased from 3 to 14 cm/sec, the gas hold-up rose from 0.2 to 3.0%, and the oxygen mass transfer coefficient, k _L a, increased from 2.5 to 18.1 h^-1. A minimum liquid velocity of 4 cm/s was required to maintain alginate gel beads (1000 m diameter, occupying 3% of reactor volume) in suspension. Batch culture of hybridoma cells immobilized in alginate beads followed logarithmic growth, reaching a concentration of 4×10⁷ cells/ml beads after 11 days. Significant antibody production did not occur until day 9 into the culture, reaching a value of 100 g/ml of medium at day 11. On the other hand, bioreactor studies with encapsulated hybridoma cells gave monoclonal antibody concentrations of up to 800 g/ml capsules (the antibody being retained within the semipermeable capsule) and maximum cell densities of 2×10⁸ cells/ml capsule at day 11. The volumetric productivities of the alginate gel immobilized cell system and the encapsulated cell system were 9 and 3 g antibody per ml of reactor volume per day, respectively. The main advantage of the bioreactor system is its simple design, since no mechanical input is required to vary the hydrodynamic properties.     

Characterization of a perfusion reactor utilizing mammalian cells on microcarrier beads

Our overall objective is to develop a cell culture analogue bioreactor (CCA) that can be used together with a corresponding physiologically based pharmacokinetic model (PBPK) to evaluate molecular mechanisms of toxicity. The PBPK is a mathematical model that divides the body into compartments representing organs, integrating the kinetic, thermodynamic, and anatomical parameters of the animal. The CCA bioreactor is a physical replica of the PBPK; where the PBPK specifies organs, the CCA bioreactor contains compartments with a corresponding cell type that mimics some of the characteristic metabolism of that organ. The device is a continuous, dynamic system composed of multiple cell types that interact through a common circulating cell culture medium. The CCA bioreactor and the model can be coupled to evaluate the plausibility of the molecular mechanism that is input into the model. This paper focuses on the design, development, and characterization of a CCA bioreactor to be used in naphthalene dose response studies. A CCA bioreactor prototype developed previously is improved upon by culturing the cells on microcarrier beads. Microcarrier beads with cells attached can form packed beds with cell culture medium perfusing the beds. In this study, two packed beds of cells, one with L2 cells (rat lung) and one with H4IIE cells (rat hepatoma), are linked in a physiologically relevant arrangement by a common recirculating cell culture medium. Studies of this CCA bioreactor presented here include mixing profiles, effect of reactor environment on cell viability and intracellular glutathione, naphthalene distribution profile, and initial naphthalene dosing studies. Unlike the prototype system there is no detectable response to naphthalene addition; in a companion paper we show that this discrepancy can be explained by differences in liquid residence times in the organ compartments. The perfusion reactor design is shown to have significant operating improvements over prototype designs.     

Ribonuclease production by free and immobilizedAspergillus clavatus cells

Several fungal strains ofAspergillus andPenicillium were immobilized by cryopolymerization in polyvinyl alcohol cryogel beads.Aspergillus clavatus was the best producer of extracellular ribonuclease. Enzyme productivity and growth of free and immobilized cells in shake flasks and agitated bioreactor were studied. Ribonuclease production and growth behaviour depended on concentrations of glucose, peptone and soybean in the culture medium. Enzyme production was influenced by agitation and aeration intensity. In repeated batch, shake-flask cultures, the immobilized cells showed 2 to 3.5 times higher enzyme activity than free cells. The optimal conditions in a bioreactor were at 150 rev/min agitation speed and 0.5 vol/vol.min aeration. Enzyme productivity of immobilized cells (237 units/g dry mycelium.h) was 2.1 times higher than the productivity of free cells in a bioreactor, and 2.3 times higher than that of a shake-flask culture.R.J. Manolov is with the Institute of Microbiology, Department of Enzymes, Bulgarian Academy of Sciences, Georgy Bonchev Street 26, 1113 Sofia, Bulgaria.     

Skeletal muscle satellite cells cultured in simulated microgravity

Summary Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and, therefore, provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (∼ 200 gm) were used for all studies and were composed of greater than 75% satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D culture and 3-D HARV culture. Plating efficiency (cells attached ÷ cells plated ×100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and nonsatellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability because glucose levels in medium from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARV were joined together by cells into 3-D aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a 3-D level of organization that could provide a more suitable model to study postnatal muscle development than is currently available with standard culture methods.     

Comparative studies on extracellular protease secretion and glucoamylase production by free and immobilized <Emphasis Type="Italic">Aspergillus niger</Emphasis> cultures

The effects of cell immobilization on the secretion of extracellular proteases and glucoamylase production by Aspergillus niger were investigated under a variety of immobilization techniques and culture conditions. Immobilization was achieved by means of cell attachment on metal surfaces or spore entrapment and subsequent growth on porous Celite beads. Free-suspension cultures were compared with immobilized mycelium under culture conditions that included growth in shake flasks and an airlift bioreactor. Cell attachment on metal surfaces minimized the secretion of proteases while enhancing glucoamylase production by the fungus. Growth on Celite beads in shake-flask cultures reduced the specific activity of the secreted proteases from 128 to 61 U g⁻¹, while glucoamylase specific activity increased from 205 to 350 U g⁻¹. The effect was more pronounced in bioreactor cultures. A reduction of six orders of magnitude in protease specific activities was observed when the fungus grew immobilized on a rolled metal screen, which served as the draft tube of an airlift bioreactor. Received 29 October 2001/ Accepted in revised form 14 June 2002     

Attachment to cytodex beads enhances differentiation of human retinal progenitors in 3-D bioreactor culture

Retinal degenerations are the leading cause of genetically inherited blindness. One of the strategies currently being tested for the treatment is cell/tissue transplantation. As such stem cells and tissue engineered constructs are of great importance. This report describes the growth of multipotential human retinal progenitors (cell line) in a 3-D bioreactor culture vessel with (adhesive substrate) laminin coated collagen 1/cytodex beads and without adhesive substrate (beadless culture). The study demonstrates that progenitors are capable of growth and differentiation in the bioreactor with or without beads. The presence of adhesive substrate accelerates and enhances photoreceptor differentiation in the bioreactor, reflected by significantly higher level expressions of several photoreceptor specific proteins; N acetyl transferase (AaNat), rhodopsin and cone transducin GNB3. Both monomeric and dimeric forms of rhodopsin are expressed in cells attached to beads, whereas, only the monomeric form is expressed in beadless culture. Similarly, a different isomeric form of tyrosine hydroxylase (a doublet) is expressed in cell bead attached cultures. Co-culturing retinal progenitors with retinal pigment epithelium (RPE) in cell bead cultures further stabilizes the photoreceptor phenotype and rhodopsin expression. Most of the retinal neuronal phenotypes are confirmed by an expression of specific proteins. The adhesive substrate in the form of collagen 1, laminin coated cytodex beads, could be just an effector for stabilization or a positive signal, modulating extracellular matrix (ECM) molecules and/or neurotrophins. In the future, the bioreactor culture system could be utilized to grow retina-like structures from ciliary epithelium by incorporating biodegradable substrates.     

Fructose production by immobilizedArthrobacter cells

Summary ImmobilizedArthrobacter cells (NRRL-B-3728) were used for continuous isomerization of glucose to fructose in a bioreactor system. The system utilized stationary phase (55h) cells (2.2×10⁹ CFU/ml saline) immobilized onto K-carrageenan (3% w/v) beads [cells were heated at 65°C for 10 min to inactivate endogenous proteolytic enzymes]. Immobilized-cell preparations were hardened using three different glutaraldehyde systems. Glutaraldehyde (0.2 M) treated-immobilized cells (pH 7.0, 5°C for 30 min) exhibited good gel strength and high glucose isomerase activities. Maximal bioreactor isomerization of 44% was achieved when a buffered feedstock containing 40% glucose was fed into the column (60°C) at a flow rate of 0.2 ml/min. The biological half-life of glucose isomerase activities in this system was 400 h. Scanning electron microscopy revealed large numbers of cells distributed within the beads. A thin layer surrounding the beads following glutaraldehyde treatment was mainly due to cross-linking reactions between cell proteins and glutaraldehyde. This layer prevented leaking of cells during continuous isomerization reaction.     

Repeated batch production of ethanol from Jerusalem artichoke tubers using recycled immobilized cells of Kluyveromyces fragilis

Summary Recycled immobilized cells of Kluyveromyces fragilis ATCC 28244 were used for repeated batch production of ethanol from the inulin sugars derived from Jerusalem artichoke tubers. Using 10% initial sugar concentration, a maximum ethanol concentration of 48 g/l was achieved in 7 h when the immobilized cell concentration in the Ca alginate beads was 72 g dry wt. immobilized cell/l bead volume. The maximum ethanol production rate was 13.5 g ethanol/l bioreactor volume/h. The same Ca alginate beads containing the cells were used repeatedly for 11 batch runs starting with fresh medium at the beginning of each run. The ethanol yield was found to be almost constant at 96% of the theoretical for all 11 batch runs, while the maximum ethanol production rate during the last batch run was found to be 70% of the original ethanol rate obtained in the first batch run.     

Semicontinuous and continuous production of penicillin-G by Penicillium chrysogenum cells immobilized in kappa-carrageenan beads

Conidia of Penicillium chrysogenum were immobilized in K-carrageenan beads and then incubated in a growth-supporting medium to yield a penicillin producing immobilized cell mass. These in situ grown immobilized cells were used for the semicontinuous (replacement cultures)and continuous (fluidized bioreactor culture) production of penicillin-G. When periodically replaced into a minimal production medium, immobilized cells exhibited a half-life for penicillin production which was ninefold greater than that exhibited by free cells. The half-life of penicillin production and the yield of penicillin from glucose in such a replacement culture were greatly affected by the frequency of replacement and by the production medium's pH and concentration of glucose, phosphate, and trace metal nutrients. A penicillin-producing continuous flow bioreactor (150 mL), employing immobilized cells, was operated for up to 16 days. The best specific penicillin productivity (1.2 mg/g cells/h)yield from glucose (7.0 mg/g glucose) and half-life of production (15 days) were obtained when the feed medium contained 10 g/L of glucose, the pH was maintained at 7.0, the relative dissolved oxygen concentration was ca. 40%; and the residence time was 20 h.     

High ethanol productivities using small Ca-alginate beads of immobilized cells of Zymomonas mobilis

Summary Zymomonas mobilis cells were immobilized into small 1 mm diameter beads of Ca-alginate in order to minimize mass transfer limitations and maximize immobilized cell activity. A combination of small bead size with a high cell concentration of 58 g dry wt. cell per lit. bead volume resulted in high ethanol productivities using a newly designed packed bed bioreactor system. Steady-state dilution rates ranging from 0.4 h^-1 to 3.9 h^-1 were run resulting in a maximum productivity of 102 g ethanol/l/h for an inlet substrate concentration of 100 g glu/l and 87% conversion. The bioreactor was run continuously at a fixed dilution rate for 384 h and short intermittent treatment of the beads with CaCl₂ temporarily increased ethanol productivity to a maximum of 116 g ethanol/l/h.     

Improvement of a fixed bed bioreactor for plant cell culture

A bioreactor used for cultivation of microbes entrapped in beads was utilized for batch culture of tobacco cells. Tobacco cells were tightly fixed on the cell-attachment net and growth rates of the cells were similar in both the bioreactor and flasks. Furthermore, there was no significant difference in the intracellular biosynthesis of phenylpropanoids between the bioreactor and flask cultures.     

Biodegradation of Phenol Using Immobilized Nocardia hydrocarbonoxydans in a Pulsed Plate Bioreactor: Effect of Packed Stages,Cell Carrier Loading,and Cell Acclimatization on Startup and Steady-State Behavior

The effect of the number of stages and cell carrier loading on the steady-state and startup performance of a continuous pulsed plate bioreactor with glass beads as the cell carrier material for biodegradation of phenol in wastewater using immobilized Nocardia hydrocarbonoxydans has been studied. It was found that the performance of the pulsed plate bioreactor during startup and at steady state can be improved by an increase in cell carrier loading, number of stages, total plate stack height, and with a decrease in plate spacing. The startup time for the continuous bioreactor can be decreased by increasing the number of preacclimatization steps for the cells. The attainment of steady effluent phenol concentration can be considered as an indication of steady state of the continuous bioreactor, as when phenol concentration attained a steady value, biofilm thickness, and the attached biomass dry weight also attained a constant value.     

Kinetics and modeling of GM-CSF production by recombinant yeast in a three-phase fluidized bed bioreactor

Continuous production of a recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) by Saccharomyces cerevisiae strain XV2181 (a/a, Trp 1) containing plasmid palphaADH2 and immobilized on porous glass beads in a fluidized bed bioreactor was studied. Kinetic models for plasmid stability, cell growth, and protein production in the three-phase fluidized bed bioreactor were developed and used to study the effects of solid loading or cell immobilization on plasmid stability and recombinant protein production. With increasing cell immobilization or solid loading in the bioreactor, plasmid stability and protein production improved significantly. The improvements could be attributed to the decreased theta value, which is the plasmid loss probability during cell division and is an indication of segregational instability of the recombinant cell, and the increased alpha value, which is the ratio of the specific growth rate of a plasmid-carrying cell to that of a plasmid-free cell and is indicative of competitive stability of the recombinant cell culture. theta decreased from 0.552 to 0.042 and alpha increased from 0.351 to 0.991 when solid loading in the bioreactor was increased from 5% (v/v) to 33%. The model simulation also showed that the specific growth rate of cells in the bioreactor was lower at higher solid loading. This indicated that there was significant mass transfer limitation, particularly for oxygen transfer, when the total cell density in the bioreactor was high at high solid loading. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 470-477, 1997.     

Cultivation of transgenicNicotiana tabacum suspension cells in bioreactors for the production of mGM-CSF

TransgenicNicotiana tabacum cells were cultivated for the production of murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in both a stirred, tank biore|actor and an airlift bioreactor with draft tube. Cell growth and mGM-CSF production in the airlift bioreactor were found to be better than those achieved in the stirred tank bioreactor. In the airlift bioreactor. 9.0 g/L of cells and 2.2 ng/mL of mGM-CSF were obtained (11.0 g/L and 2.4 ng/mL, respectively in shake flasks). Although the lag period was prolonged and mGM-CSF production was lowered by 33% in the stirred tank bioreactor as compared to the control culture, the maximum cell density was increased up to 12.0 g/L due to better mixing by agitation at the higher cell density.     

Production of Superoxide Dismutase from Streptococcus lactis Using a Bioreactor with a Microfiltration Module

The performance of a bioreactor with a microfiltration module for the production of an intracellular enzyme, superoxide dismutase (SOD), by Streptococcus lactis is described. The fermentation system involving the bioreactor enables the continuous removal of metabolites inhibitory for cell growth and the complete recycling of the cells to the bioreactor. In a fed-batch (FB) culture with filtration, in which the main metabolite, lactic acid, in the culture broth was maintained at a low concentration, S. lactis was cultivated to the high concentration of 15.5 g-dry cells/1. The SOD content of the cells remained at almost a constant level throughout the cultivation and the productivity of SOD as well as cells per unit time was 4.3-fold as high as that in the case of a conventional batch culture without filtration. Repeating the FB culture with filtration enhanced the productivities of SOD and cells further, as compared with those in the case of the FB culture with filtration.     

Culturing of primary hepatocytes as entrapped aggregates in a packed bed bioreactor: A potential bioartificial liver

Summary Conventional culture systems for hepatocytes generally involve cells cultured as flat, monolayer cells, with limited cell-cell contact, in a static pool of medium, unlike the liver in vivo where the parenchymal cells are cuboidal, with extensive cell-cell contact, and are continuously perfused with blood. We report here a novel bioreactor system for the culturing of primary hepatocytes with cuboidal cell shape, extensive cell-cell contact, and perfusing medium. The hepatocytes were inoculated into the bioreactor and allowed to recirculate at a rate optimal for them to collide and form aggregates. These newly-formed aggregates were subsequently entrapped in a packed bed of glass beads. The bioreactor was perfused with oxygenated nutrient medium, with controlled oxygen tension, pH, and medium perfusion rate. The hepatocytes were viable for up to the longest time point studied of 15 days in culture based on urea synthesis, albumin synthesis and cell morphology. Light microscopy studies of hepatocytes cultured for 15 days in the bioreactor showed interconnecting three-dimensional structures resembling the hepatic cell plate in the liver organ. Electron microscopy studies on the same cells revealed ultrastructure similar to the hepatocytes in vivo, including the presence of plentiful mitochondria, rough and smooth endoplasmic reticulum, glycogen granules, peroxisomes, and desmosomes. We believe that our hepatocyte bioreactor is a major improvement over conventional culture systems, with important industrial applications including toxicology, drug metabolism, and protein/peptide synthesis. The hepatocyte bioreactor concept may also be used as the basis for the development of a bioartificial liver to provide extracorporeal hepatic support to patients with hepatic failure.     

Phytate degradation by immobilized<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> phytase in soybean-curd whey

Saccharomyces cerevisiae CY phytase-producing cells were immobilized in calcium alginate beads and used for the degradation of phylate. The maximum activity and immobilization yield of the immobilized phytase reached 280 mU/g-bead and 43%, respectively. The optimal pH of the immobilized cell phytase was not different from that of the free cells. However, the optimum temperature for the immobilized phytase was 50°C, which was 10°C higher than that of the free cells; pH and thermal stability were enhanced as a consequence of immobilization. Using the immobilized phytase, phytate was degraded in a stirred tank bioreactor. Phytate degradation, both in a buffer solution and in soybean-curd whey mixture, showed very similar trends. At an enzyme dosage of 93.9 mU/g-phytate, half of the phytate was degraded after 1 h of hydrolysis. The operational stability of the immobilized beads was examined with repeated batchwise operations. Based on 50% conversion of the phytate and five times of reuse of the immobilized beads, the specific degradation (g phytate/g dry cell weight) for the immobilized phytase increased 170% compared to that of the free phytase.     

Improved oxygen transfer and increased l-lactic acid production by morphology control of Rhizopus oryzae in a static bed bioreactor

Rhizopus oryzae was immobilized on a cotton matrix in a static bed bioreactor. Compared with free cells in a stirred tank bioreactor, immobilized R. oryzae in this bioreactor gave higher lactic acid production but lower ethanol production. The highest lactic acid production rate (2.09 g/L h) with the final concentration of 37.83 g/L from 70 g/L glucose was achieved when operating the bioreactor at 700 rpm and 0.5 vvm air. To better understand the relationship between shear effects (agitation and aeration) and R. oryzae morphology and metabolism, oxygen transfer rate, fermentation kinetics, and lactate dehydrogenase activity were determined. In immobilized cell culture, higher oxygen transfer rate and lactic acid production were achieved but lower lactate dehydrogenase activity was found as compared with those in free cell culture operated at the same conditions. These results clearly imply that mass transport was the rate controlling step in lactic acid fermentation by R. oryzae.