Effect of oxygen on the growth, respiratory rate and morphology of Candida utilis cells in periodic and continuous cultures

The aim of this work was to study how the concentration of oxygen dissolved in the cultural broth influenced the respiration and morphology of the yeast Candida utilis in batch and continuous cultures. Highly effective respiration was registered in cells growing for a certain period of time at low oxygen concentrations limiting the growth; the respiration was characterized by low values of the Michaelis constant kc and the critical concentration of dissolved oxygen Ccr. When passing from the low oxygen concentration to a high one, the character of cellular respiration changed abruptly in the cells whose growth was limited with oxygen for a long time. The morphology of the culture limited with oxygen was characterized by an increase in the percentage of elongated forms in the population. The respiration of the cells cultivated at high oxygen concentrations, when their growth was either non-limited or limited by glucose, was distinguished by high Ccr values and slow respiration rates at small oxygen concentrations while the dependence of the respiration rate on the concentration of oxygen had an about S-shaped character.     

Measurement of low levels of oxygen and their effect on respiration in cell suspensions maintained in an open system

The presence of low levels of oxygen may have profound effects on the cytotoxic activity of radiation, radiosensitizers, and bioreductive alkylating agents. As others have shown, low oxygen tensions may significantly alter rates of cellular and chemical oxygen consumption. When experiments are performed at very low oxygen concentrations, the opposing effects of oxygen leakage into and cellular/chemical oxygen consumption from the system can lead to unpredictable results. Use of a newly designed, highly sensitive Clark-type oxygen sensor has permitted accurate and reproducible measurement of low levels of oxygen. Cellular depletion of oxygen at various cell densities has been monitored for a series of oxygen tensions in solution and the corresponding respiration rates have been calculated. Although oxygen depletion was found to be quite significant at low oxygen tensions, not all oxygen present could be removed by cellular respiration. Respiration rate decreased as oxygen tension decreased and approached zero at low oxygen tensions. This result was independent of cell density. A model is presented to account for the observed effect of oxygen tension on cellular oxygen utilization.     

Positive effects of proline addition on the central metabolism of wild-type and lactic acid-producing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains

In Saccharomyces cerevisiae, proline is a stress protectant interacting with other substrate uptake systems against oxidative stress under low pH conditions. In this study, we performed metabolomics analysis to investigate the response associated with an increase in cell growth rates and maximum densities when cells were treated with proline under normal and acid stress conditions. Metabolome data show that concentrations of components of central metabolism are increased in proline-treated S. cerevisiae. No consumption of proline was observed, suggesting that proline does not act as a nutrient but regulates metabolic state and growth of cells. Treatment of lactic acid-producing yeast with proline during lactic acid bio-production improved growth rate and increased the final concentration of lactic acid.     

High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficients were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric realations for dynamic nutrient feeding in high cell concentration fed batch cultures. (c) 1995 John Wiley & Sons, Inc.     

High cell density suspension culture of mammalian anchorage independent cells: Oxygen transfer by gas sparging and defoaming with a hydrophobic net

Gas sparging directly into the culture-broth is not done in cell culture, except when the gas flow rate is very small, because much foaming occurs.During screening of defoaming methods, foam was observed to be broken up effectively when it made contact with a net fabricated from hydrophobic materials. Providing a highly efficient oxygen supply to suspension culture was tried using the new defoaming method. In a 5 1 reactor equipped with the foam-eliminating net fabricated with polysiloxane, oxygen was transferred at 21 mmole/l·h equivalent to an about forty-fold higher rate than in conventional surface aeration. This was equivalent to a consumption rate of 1×10⁸ cells/ml, even at a low oxygen gas flow rate of 0.1 cm/s corresponding to a fourth of the gas flow rate when foam leaked through the net.Perfusion culture of rat ascites hepatoma cell JTC-1 was successfully carried out in the 51 scale culture system with the net and a hydrophobic membrane for cell filtration. The viable cell concentration reached 2.7×10⁷ cells/ml after twenty-seven days, in spite of the nutrient-deficient condition of the lower medium exchange rate, that is, a working volume a day, and viability was maintained at more than 90%. In a 1.21 scale culture of mouse-mouse hybridoma cell STK-1, viable cell concentration reached 4×10⁷ cells/ml. These results showed that oxygen transfer by gas sparging with defoaming was useful for high density suspension culture. A foam-breaking mechanism was proposed.Abbreviations Eagle's MEM Eagle's minimal essential medium - Dulbecco's modified Eagle MEM Dulbecco's modified Eagle minimal essential medium     

Mitochondrial hydrogen peroxide production alters oxygen consumption in an oxygen-concentration-dependent manner

Metabolic responses of mammalian cells toward declining oxygen concentration are generally thought to occur when oxygen limits mitochondrial ATP production. However, at oxygen concentrations markedly above those limiting to mitochondria, several mammalian cell types display reduced rates of oxygen consumption without energy stress or compensatory increases in glycolytic ATP production. We used mammalian Jurkat T cells as a model system to identify mechanisms responsible for these changes in metabolic rate. Oxygen consumption was 31% greater at high oxygen (150–200 μM) compared to low oxygen (5–10 μM). Hydrogen peroxide was implicated in the response as catalase prevented the increase in oxygen consumption normally associated with high oxygen. Cell-derived hydrogen peroxide, predominately from the mitochondria, was elevated with high oxygen. Oxygen consumption related to intracellular calcium turnover was shown, through EDTA chelation and dantrolene antagonism of the ryanodine receptor, to account for 70% of the response. Oligomycin inhibition of oxygen consumption indicated that mitochondrial proton leak was also sensitive to changes in oxygen concentration. Our results point toward a mechanism in which changes in oxygen concentration influence the rate of hydrogen peroxide production by mitochondria, which, in turn, alters cellular ATP use associated with intracellular calcium turnover and energy wastage through mitochondrial proton leak.     

Role of nutrient supply on cell growth in bioreactor design for tissue engineering of hematopoietic cells

In the present study, a dynamic mathematical model for the growth of granulocyte progenitor cells in the hematopoietic process is developed based on the principles of diffusion and chemical reaction. This model simulates granulocyte progenitor cell growth and oxygen consumption in a three-dimensional (3-D) perfusion bioreactor. Material balances on cells are coupled to the nutrient balances in 3-D matrices to determine the effects of transport limitations on cell growth. The method of volume averaging is used to formulate the material balances for the cells and the nutrients in the porous matrix containing the cells. All model parameters are obtained from the literature. The maximum cell volume fraction reached when oxygen is depleted in the cell layer at 15 days and is nearly 0.63, corresponding to a cell density of 2.25 x 10(8) cells/mL. The substrate inhibition kinetics for cell growth lead to complex effects with respect to the roles of oxygen concentration and supply by convection and diffusion on cell growth. Variation in the height of the liquid layer above the cell matrix where nutrient supply is introduced affected the relative and absolute amounts of oxygen supply by hydrodynamic flow and by diffusion across a gas permeable FEP membrane. Mass transfer restrictions of the FEP membrane are considerable, and the supply of oxygen by convection is essential to achieve higher levels of cell growth. A maximum growth rate occurs at a specific flow rate. For flow rates higher than this optimal, the high oxygen concentration led to growth inhibition and for lower flow rates growth limitations occur due to insufficient oxygen supply. Because of the nonlinear effects of the autocatalytic substrate inhibition growth kinetics coupled to the convective transport, the rate of growth at this optimal flow rate is higher than that in a corresponding well-mixed reactor where oxygen concentration is set at the maximum indicated by the inhibitory kinetics.     

Variations in tumor cell growth rates and metabolism with oxygen concentration, glucose concentration, and extracellular pH.

Tumors and multicellular tumor spheroids can develop gradients in oxygen concentration, glucose concentration, and extracellular pH as they grow. In order to calculate these gradients and assess their impact on tumor growth, it is necessary to quantify the effect of these variables on tumor cell metabolism and growth. In this work, the oxygen consumption rates, glucose consumption rates, and growth rates of EMT6/Ro mouse mammary tumor cells were measured at a variety of oxygen concentrations, glucose concentrations, and extracellular pH levels. At an extracellular pH of 7.25, the oxygen consumption rate of EMT6/Ro cells increased by nearly a factor of 2 as the glucose concentration was decreased from 5.5 mM to 0.4 mM. This effect of glucose concentration on oxygen consumption rate, however, was slight at an extracellular pH of 6.95 and disappeared completely at an extracellular pH of 6.60. The glucose consumption rate of EMT6/Ro cells increased by roughly 40% when the oxygen concentration was reduced from 0.21 mM to 0.023 mM and decreased by roughly 60% when the extracellular pH was decreased from 7.25 to 6.95. The growth rate of EMT6/Ro cells decreased with decreasing oxygen concentration and extracellular pH; however, severe conditions were required to stop cell growth (0.0082 mM oxygen and an extracellular pH of 6.60). Empirical correlations were developed from these data to express EMT6/Ro cell growth rates, oxygen consumption rates, and glucose consumption rates, as functions of oxygen concentration, glucose concentration, and extracellular pH. These empirical correlations make it possible to mathematically model the gradients in oxygen concentration, glucose concentration, and extracellular pH in EMT6/Ro multicellular spheroids by solution of the diffusion/reaction equations. Computations such as these, along with oxygen and pH microelectrode measurements in EMT6/Ro multicellular spheroids, indicated that nutrient concentration and pH levels in the inner regions of spheroids were low enough to cause significant changes in nutrient consumption rates and cell growth rates. However, pH and oxygen concentrations measured or calculated in EMT6/Ro spheroids where quiescent cells have been observed were not low enough to cause the cessation of cell growth, indicating that the observed quiescence must have been due to factors other than acidic pH, oxygen depletion, or glucose depletion.     

Design of a bubble-swawm bioreactor for animal cell culture

A stationary bubble-swarm has been used to aerate a mammalian cell culture bioreactor with an extremely low gas flow rate. Prolonging the residence time of the gas bubbles within the medium improved the efficiency of the gas transfer into the liquid phase and suppressed foam formation. An appropriate field of speed gradients prevented the bubbles from rising to the surface. This aeration method achieves an almost 90% transfer of oxygen supplied by the bubbles. Consequently, it is able to supply cells with oxygen even at high cell densities, while sparging with a gas flow of only 0.22·10^–3–1.45·10^–3 vvm (30–200 ml/h).The reactor design, the oxygen transfer rates and the high efficiency of the system are presented. Two repeated batch cultures of a rat-mouse hybridoma cell line are compared with a surface-aerated spinner culture. The used cell culture medium was serum-free, either with or without BSA and did not contain surfactants or other cell protecting agents. One batch is discussed in detail for oxygen supply, amino acid consumption and specific antibody production.     

Stability studies of recombinant Saccharomyces cerevisiae in the presence of varying selection pressure

A recombinant yeast plasmid carrying the Ieu2 gene for auxotrophic complementation and a reporter gene for beta-galactosidase under the control of Gal10 promoter was studied in Saccharomyces cerevisiae. Growth, product formation, and plasmid stability were studied in defined, semi-defined, and complex media. The biomass concentration and specific activity were higher in complex medium than in defined medium, which was selective for the growth of plasmid-containing cells, leading to a 10-fold increase in volumetric activity. However, plasmid instability was very high in complex media with 50% plasmid-free cells emerging in the culture within 75 h of cultivation. In order to control instability, the growth rates of the plasmid-containing and plasmid-free cells were determined in semi-defined media, which consisted of defined medium supplemented with different concentrations of yeast extract. Below a critical concentration of yeast extract (0.05 g/L), the plasmid-containing cells had a growth rate advantage over the plasmid-free cells. This was possibly because, at this concentration of yeast extract, the availability of leucine became the rate-determining factor in the specific growth rate of plasmid-free cells. A feeding strategy was designed which maintained a low concentration of the residual yeast extract in the medium and thus continuously provided the plasmid-containing cells with a competitive advantage over the plasmid-free cells. This resulted in high stability as well as high cell density under non-selective conditions, which led to a 10-fold increase in the volumetric activity compared to that achieved in defined selective media. A simple mathematical model was formulated to verify the experimental data. The important state variables and process parameters, i.e., biomass concentration, beta-galactosidase expression, sucrose consumption, yeast extract consumption, and specific growth rates of the two cell populations, were evaluated. These variables and parameters along with the differential equations based on material balances as well as the experimental results obtained were used in a mathematical model for the fed-batch cultivation. These correctly verified the experimental data and clearly illustrated the concept behind the success of the fed-batch strategy under yeast extract starvation.     

Acetate metabolism by Escherichia coli in high-cell-density fermentation.

Little is known about the cellular physiology of Escherichia coli at high cell densities (e.g., greater than 50 g [dry cell weight] per liter), particularly in relation to the cellular response to different growth conditions. E. coli W3100 cultures were grown under identical physical and nutritional conditions, by using a computer-controlled fermentation system which maintains the glucose concentration at 0.5 g/liter, to high cell densities at pH values of 6.0, 6.5, 7.0, and 7.5. The data suggest a relationship between the pH of the environment and the amount of acetate excreted by the organism during growth. At pH values of 6.0 and 6.5, the acetate reached a concentration of 6 g/liter, whereas at pH 7.5, the acetate reached a concentration of 12 g/liter. Furthermore, at pH values of 6.0 to 7.0, the E. coli culture undergoes a dramatic metabolic switch in which oxygen and glucose consumption and CO2 evolution all temporarily decreased by 50 to 80%, with a concomitant initiation of acetate utilization. After a 30-min pause in which approximately 50% of the available acetate is consumed, the culture recovers and resumes consuming glucose and oxygen and producing acetate and CO2 at preswitch levels. During the switch period, the specific activity of isocitrate lyase typically increases approximately fourfold.     

Protective effect of antioxidants against para-nonylphenol-induced inhibition of cell growth in Saccharomyces cerevisiae

The cell growth-modulating activity of an endocrine disruptor, p-nonylphenol (NP), was estimated using the yeast Saccharomyces cerevisiae as a simple model of eukaryotic cells. NP caused a dose-dependent suppressive effect on cell growth of S. cerevisiae at 10, 25 and 50 microM. The NP-induced cell growth inhibition was restored when concomitantly lipophilic antioxidants such as alpha-tocopherol and beta-carotene were supplied, but not the hydrophilic antioxidants ascorbic acid or (-)epigallocatechin gallate (EGCG). The cellular oxygen consumption of S. cerevisiae was also inhibited in a dose-dependent fashion by the extracellular addition of NP, and pretreatment with alpha-tocopherol and beta-carotene suppressed NP-induced inhibition of cellular oxygen consumption, but ascorbic acid and EGCG were not effective. Furthermore, NP caused a marked generation of radical oxygen species (ROS) in S. cerevisiae, which was suppressed by treatment with alpha-tocopherol and beta-carotene, but not with ascorbic acid and EGCG. However, NP did not show a significant inhibitory effect on cell growth and survival of mitochondria-deficient petite mutant cells and they showed a relatively weak ROS-generating activity compared with parent yeast cells. These results suggest that NP-induced inhibition of cell growth and oxygen consumption in S. cerevisiae might be possibly associated with ROS generation in yeast mitochondria. The significance of this finding is discussed from the viewpoint of NP-induced oxidative stress against eukaryotic cells.     

Biochemical study of idebenone effect on mitochondrial metabolism of yeast

The aim of this work was to study the effect of the drug idebenone on the growth of a strain of Saccharomyces cerevisiae yeast and its respiratory-deficient mutant (rho(0)). We took this yeast as a model system of the interaction of the drug with mammalian cells. The effect of idebenone was evaluated in rich and minimal media. In the S288c strain, idebenone exerted a growth inhibitory effect in concentrations higher than 50 microM in media containing a carbon source consumed at mitochondrial level. In conditions of low oxygen supply, idebenone allows yeast to keep a cellular yielding comparable with conditions of normal oxygen supply. Also, the presence of idebenone in the growth media increased by 50% the fluorescence signal of rhodamine 123, indicating a higher mitochondrial membrane potential. The results could explain the effect of idebenone in the treatment of diseases in which oxygen deficiency alters the energetic metabolism of the cell.     

Hybridoma growth and antibody production as a function of cell density and specific growth rate in perfusion culture

Steady state metabolic parameters for hybridoma cell line H22 were determined over a wide range of cell densities and specific growth rates in a filtration based homogeneous perfusion reactor. Operating the reactor at perfusion rates of 0.75, 2.0, and 2.9 day(-1)(each at four different specific growth rates), viable cell densities as high as 2 x 10(7) cells/mL were obtained. For the cell line under investigation, the specific monoclonal antibody production rate was found to be a strong function of the viable cell density, increasing with increasing cell density. In contrast, most of the substrate consumption and product formation rates were strong functions of the specific growth rate. Substrate metabolism became more efficient at high cell densities and low specific growth rates. The Specific rates of metabolite formation and the apparent yields of lactate from glucose and ammonia from glutamine decreased at low specific growth rates and high cell densities. While the specific oxygen consumption rate was independent of the specific growth rate and cell density, ATP production was more oxidative at lower specific growth rate and higher cell density. These observed shifts are strong indications of the production potential of high-density perfusion culture. (c) 1995 John Wiley & Sons, Inc.     

Characterization of changes in PER.C6 cellular metabolism during growth and propagation of a replication-deficient adenovirus vector

PER.C6 cells were cultivated for propagation of a replication-defective adenovirus vector in serum-free suspension bioreactors. Cellular metabolism during cell growth and adenovirus propagation was fully characterized using on-line and off-line methods. The energy metabolism was found to accelerate transiently after adenovirus infection with increases in glucose and oxygen consumption rates. Similar to other mammalian cells, glucose utilization was highly inefficient and a high lactate:glucose yield was observed, both before and after virus infection. A higher consumption of most of the essential amino acids was observed transiently after the infection, likely due to increased protein synthesis requirements for virus propagation. To improve virus propagation, a medium exchange strategy was implemented to increase PER.C6 cell concentration for infection. During cell growth, a 50% increase in glucose consumption and lactate production rates was observed after initiation of the medium exchange in comparison to the batch phase. This decrease in medium capacity only affected the central carbon metabolism and no increase in amino acid consumption was observed. In addition, even though cell concentrations of up to 10 x 10(6) cells/mL were reproducibly obtained by medium exchange, infections at cell concentrations higher than 1 x 10(6) cells/mL did not proportionally improve volumetric adenovirus productivities. No measured nutrient limitation was observed at those high cell concentrations, indicating that adenovirus cell-specific productivity at higher cell concentrations is highly dependent on cell physiology. These results provide a better understanding of PER.C6 cellular metabolism and a basis for intensifying PER.C6 growth and adenovirus propagation.     

CHO-K1 host cells adapted to growth in glutamine-free medium by FACS-assisted evolution

During the process of recombinant cell line optimisation for production of biopharmaceuticals, multiple cellular properties like robustness against stress, the attainment of high cell concentrations and maintenance of high viability must be considered to maximize protein yield. To improve growth and viability, glutamine is supplemented as an alternative energy source for rapidly dividing cells that oxidize glucose inefficiently. However, the resulting by-product ammonia is toxic at high concentrations and has a negative impact on protein glycosylation, a major quality-determining parameter of biopharmaceuticals. In this work, the CHO-K1 cell line was adapted to a chemically defined medium and suspension growth within 3 weeks. Subsequently, the glutamine concentration was stepwise reduced from 8 to 4 and 2 mM. After each reduction, both the final cell concentration in the batch and the viability decreased. To force a rapid evolution of cells to achieve high final cell concentrations, cells were seeded at high densities (10(7) cells/mL) and surviving cells were sorted by FACS or MACS when viability declined to 10% (typically after 24 h). Sorted cells were grown in batch until viability declined to 10% and viable cells recovered again. The final sorted population was able to reach comparable or even better viable cell concentrations and showed a significantly improved viability compared to their ancestors. The 2 mM glutamine-adapted cell line was directly transferred into glutamine-free medium and was able to grow at comparable rates without requiring further adaptation. Cells compensated the lack of glutamine by increasing their consumption of glutamate and aspartate.     

Kinetic modelling of continuous submerged fermentation of cheese whey for single cell protein production

A mathematical model describing the kinetics of continuous production of single cell protein from cheese whey using Kluyveromyces fragilis was developed from the basic principles of mass balance. The model takes into account the substrate utilization for growth and maintenance and the effect of substrate concentration and cell death rate on the net cell growth and substrate utilization during the fermentation process. A lactose concentration below 1.91 g/L limited growth of yeast cells whereas a lactose concentration above 75 g/L inhibited the growth of the yeast. The model was tested using experimental data obtained from a continuous system operated at various retention times (12, 18 and 24 h), mixing speeds (200, 400 and 600 rpm) and air flow rates (1 and 3 vvm). The model was capable of predicting the effluent cell and substrate concentrations with R2 ranging from 0.95 to 0.99. The viable cell mass and lactose consumption ranged from 1.3 to 34.3 g/L and from 74.31% to 99.02%, respectively. A cell yield of 0.74 g cell/g lactose (close to the stoichiometric value of 0.79 g cell/g lactose) was achieved at the 12 h retention time-3 vvm air flow rate-600 rpm mixing speed combination. The total biomass output (viable and dead cells) at this combination was 37 g/L.     

The effect of oxygen tension on haemopoietic and fibroblast cell proliferation in vitro

The effects of providing low oxygen tension in the gas phase of two different types of cell culture systems were investigated. The clonal growth of granulocyte-macrophage progenitor cells in an agar culture system was improved markedly by incubation within a low oxygen tension gas phase (48 mmHg – 6.8%) instead of the conventional air (135 mmHg – 19%), the effects being measured by increases in numbers of colony forming cells detected and in the colony sizes. The increased efficiency of colony formation was observed both with mouse and human marrow cells. A similar effect was observed in a liquid adherence culture system with primary cultures of foetal mouse fibroblasts both at clonal and higher cell densities.     

The effect of dissolved oxygen and aeration rate on antibiotic production of Streptomyces fradiae

Different dissolved oxygen concentrations and aeration rates were imposed on a stable mutant of Streptomyces fradiae during the antibiotic-producing phase. At high aeration rate (1 vvm), the tylosin yield in the fermentor broth with dissolved oxygen (DO) concentrations controlled close to 100% saturation (6-8 ppm) increased 10% as against uncontrolled. The rates of cellular growth, oil consumption, and tylosin production were severely reduced when DO concentration fell below 25% saturation, but all resumed to their initial rates when DO was raised to saturation level again. The DO concentration in combination with air flow rate affected the pattern of the antibiotics produced. At high DO levels, an additional macrolide antibiotic, macrocin, was synthesized to more than one-third the amount of tylosin at high aeration rate (1 vvm). On the other hand, tylosin production rate remained constant and no significant amount of macrocin was produced at low aeration rate (0.2 vvm).     

Dependence of the effect of RNAase from Bacillus intermedius on the growth of baker's yeast on the concentration of the exogenous enzyme

Bacillus intermedius RNase added at a low concentration (0.001 microgram/ml) stimulated yeast growth, while a high RNase concentration (1500 micrograms/ml) was inhibitory to yeast growth. The inhibitory effect of RNase was transient and correlated with the increase in the trehalose pool of yeast cells. The number of unbudded cells in the yeast population tended to decrease under the action of low concentrations of bacillar RNase and to increase under the action of high concentrations of this enzyme.