Growth characteristics of channel catfish ovary cells—influence of glucose and glutamine

The growth characteristics and influence of glucose and glutamine on the growth and maintenance of channel catfish ovary (CCO) cells were investigated. Besides glutamine, amino acids threonine, arginine, methionine and serine were found to be essential for CCO cell growth. In the glucose-free medium, glutamine is utilized as energy source and no cell growth limitation was observed. However, the lack of glutamine in culture medium did not stimulate CCO cells to efficient glucose consumption. When both glucose and glutamine were deficient, cell growth was also observed suggesting no rigorous nutritional requirements. Obtained results are useful for further understanding of culture processes using CCO cells.     

Stoichiometry,Kinetics, and Regulation of Glucose and Amino Acid Metabolism of a Recombinant BHK Cell Line in Batch and Continuous Cultures

Batch and continuous cultures were carried out to study the stoichiometry, kinetics, and regulation of glucose and amino acid metabolism of a recombinant BHK cell line, with particular attention to the metabolism at low levels of glucose and glutamine. The apparent yields of cells on glucose and glutamine, lactate on glucose, and ammonium on glutamine were all found to change significantly at low residual concentrations of glucose (<5 mmol/L) and glutamine (<1 mmol/L) . The uptake rates of glucose and glutamine were markedly reduced at low concentrations, leading to a more effective utilization of these nutrients for energy metabolism and biosynthesis and reduced formation rates of lactate and ammonium. However, the consumption of other amino acids, especially the essential amino acids leucine, isoleucine, and valine and the nonessential amino acids serine and glutamate, was strongly enhanced at low glutamine concentration. Quantitatively, it was shown that the cellular yields and rates associated with glucose metabolism were primarily determined by the residual glucose concentration, while those associated with glutamine metabolism depended mainly on the residual glutamine. Both experimental results and analysis of the kinetic data with models showed that the glucose metabolism of BHK cells is not affected by glutamine except for a slight influence under glucose limitation and glutaminolysis not by glucose, at least not significantly under the experimental conditions. Compared to hybridoma and other cultured animal cells, the recombinant BHK cell line showed remarkable differences in terms of nutrient sensitivity, stoichiometry, and amino acid metabolism at low levels of nutrients. These cell-line-specific stoichiometry and nutrient needs should be considered when designing an optimal medium and/or feeding strategy for achieving high cell density and high productivity of BHK cells. In this work, a cell density of 1.1 × 10⁷ cells/mL was achieved in a conventional continuous culture by using a proper feed medium.     

Development of a fed-batch cultivation for antibody-producing cells based on combined feeding strategy of glucose and galactose

In order to achieve enhanced cell mass and productivity with less lactate accumulation, a fed-batch culture based on a combined feeding strategy of glucose and galactose was developed. Cell performance was first examined with feeding of galactose alone. While cell growth was improved compared with glucose-feeding culture, cell maintenance was inefficient with rapid lactate depletion and considerable ammonium accumulation. Subsequently, to improve cell maintenance, a combined feeding strategy of glucose and galactose was proposed focusing on optimizing the ratio of glucose to galactose and feeding time. In addition, the compositions of amino acids and vitamins in feeding medium were refined for balanced supply of nutrients. With the combined feeding strategy, the metabolic shift of lactate from production to consumption occurred, but not accompanied by rapid lactate depletion and ammonium production. Furthermore, energy metabolism was more efficient and better utilization of carbon sources was achieved. Compared with the glucose-feeding culture in bioreactor, maximum lactate concentration was reduced by 55%; IVCC and the specific production rate of antibody were increased by 45% and 143%, respectively.     

Serum-free suspension cultivation of PER.C6(R) cells and recombinant adenovirus production under different pH conditions

PER.C6(R) cell growth, metabolism, and adenovirus production were studied in head-to-head comparisons in stirred bioreactors under different pH conditions. Cell growth rate was found to be similar in the pH range of 7.1-7.6, while a long lag phase and a slower growth rate were observed at pH 6.8. The specific consumption rates of glucose and glutamine decreased rapidly over time during batch cell growth, as did the specific lactate and ammonium production rates. Cell metabolism in both infected and uninfected cultures was very sensitive to culture pH, resulting in dramatic differences in glucose/glutamine consumption and lactate/ammonium production under different pH conditions. It appeared that glucose metabolism was suppressed at low pH but the efficiency of energy production from glucose was enhanced. Adenovirus infection resulted in profound changes in cell growth and metabolism. Cell growth was largely arrested under all pH conditions, while glucose consumption and lactate production were elevated post virus infection. Virus infection induced a reduction in glutamine consumption at low pH but an increase at high pH. The optimal pH for adenovirus production was found to be 7.3 under the experimental conditions used in the study. Deviations from this optimum resulted in significant reductions of virus productivity. The results indicate that culture pH is a very critical process parameter in PER.C6(R) cell culture and adenovirus production.     

Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production

The influence of ammonia and lactate on cell growth, metabolic, and antibody production rates was investigated for murine hybridoma cell line 163.4G5.3 during batch culture. The specific growth rate was reduced by one-half in the presence of an initial ammonia concentration of 4 mM. Increasing ammonia levels accelerated glucose and glutamine consumption, decreased ammonia yield from glutamine, and increased alanine yield from glutamine. Although the amount of antibody produced decreased with increasing ammonia concentration, the specific antibody productivity remained relatively constant around a value of 0.22 pg/cell-h. The specific growth rate was reduced by one-half at an initial lactate concentration of 55 mM. Although specific glucose and glutamine uptake rates were increased at high lacatate concentration, they showed a decrease after making corrections for medium osmolarity. The yield coefficient of lactate from glucose decreased at high lactate concentrations. A similar decrease was observed for the ammonia yield coefficient from glutamine. At elevated lactate concentrations, specific antibody productivities increased, possibly due to the increase in medium osmolarity. The specific oxygen uptake rate was insensitive to ammonia and lactate concentrations. Addition of ammonia and lactate increased the calculated metabolic energy production of the cells. At high ammonia and lactate, the contribution of glycolysis to total energy production increased. Decreasing external pH and increasing ammonia concentrations caused cytoplasmic acidification. Effect of lactate on intracellular pH was insignificant, whereas increasing osmolarity caused cytoplasmic alkalinization.     

Glutamine dependency of human skin fibroblasts: modulation by hexoses

The combined effects of carbohydrates and glutamine were investigated in diploid strains of normal human skin fibroblasts cultured for 21 days under eight different culture conditions: hexose-free medium or medium containing D-glucose, D-galactose, or D-fructose, with or without added glutamine. Cell growth, hexose consumption, lactate production, intracellular glycogen content and extracellular amino acid levels were measured every third to fourth day. In the presence of glutamine, cells reached a higher saturation density in fructose medium than in glucose or galactose medium but per cell consumption of fructose and galactose was much less than that of glucose. Consumption of all three carbohydrates per unit cell growth exhibited three distinct phases: Days 1-3, 3-10, and 10-20, respectively. In the absence of glutamine the rate of cell growth was not altered in glucose or galactose medium, but slowed down considerably in fructose medium. Glutamine deprivation also led to changes in hexose consumption. In hexose-free media the cell growth rate at first was very slow, but rose after 2 or 3 weeks of culture. The levels of extracellular nonessential amino acids varied according to medium and growth phase. One of the most exciting findings was that human fibroblasts are able to maintain a slight excess of glutamine in all media not supplemented with glutamine and, more surprisingly, to synthesize it in a medium containing galactose and glutamine.     

Metabolism of PER.C6 cells cultivated under fed-batch conditions at low glucose and glutamine levels

This is the first study to examine PER.C6 cell glucose/energy and glutamine metabolism with fed-batch cultures at controlled low glutamine, low glucose, and simultaneous low glucose and low glutamine levels. PER.C6(TM) cell metabolism was investigated in serum-free suspension bioreactors at two-liter scale. Control of glucose and/or glutamine concentrations had a significant effect on cellular metabolism leading to an increased efficiency of nutrient utilization, altered byproduct synthesis, while having no effect on cell growth rate. Cultivating cells at a controlled glutamine concentration of 0.25 mM reduced q(Gln) and q(NH(4)(+)) by approximately 30%, q(Ala) 85%, and q(NEAA) 50%. The fed-batch control of glutamine also reduced the overall accumulation of ammonium ion by approximately 50% by minimizing the spontaneous chemical degradation of glutamine. No major impact upon glucose/energy metabolism was observed. Cultivating cells at a glucose concentration of 0.5 mM reduced q(Glc) about 50% and eliminated lactate accumulation. Cells exhibited a fully oxidative metabolism with Y(O(2)/Glc) of approximately 6 mol/mol. However, despite no increase in q(Gln), an increased ammonium ion accumulation and Y(NH(4)(+)/Gln) were also observed. Effective control of lactate and ammonium ion accumulation by PER.C6 cells was achieved using fed-batch with simultaneously controlled glucose and glutamine. A fully oxidative glucose metabolism and a complete elimination of lactate production were obtained. The q(Gln) value was again reduced and, despite an increased q(NH(4)(+)) compared with batch culture, ammonium ion levels were typically lower than corresponding ones in batch cultures, and the accumulation of non-essential amino acids (NEAA) was reduced about 50%. In conclusion, this study shows that PER.C6 cell metabolism can be confined to a state with improved efficiencies of nutrient utilization by cultivating cells in fed-batch at millimolar controlled levels of glucose and glutamine. In addition, PER.C6 cells fall into a minority category of mammalian cell lines for which glutamine plays a minor role in energy metabolism.     

Catabolic control of hybridoma cells by glucose and glutamine limited fed batch cultures

Substrate limited fed batch cultures were used to study growth and overflow metabolism in hybridoma cells. A glucose limited fed batch, a glutamine limited fed batch, and a combined glucose and glutamine limited red batch culture were compared with batch cultures. In all cultures mu reaches its maximum early during growth and decreases thereafter so that no exponential growth and decreases thereafter so that no exponential growth rate limiting, although the glutamine concentration (>0.085mM) was lower than reported K(s) vales and glucose was below 0.9mM; but some other nutrients (s) was the cause as verified by simulations. Slightly more cells and antibodies were produced in the combined fed batch compared with the batch culture. The specific rates for consumption of glucose and glutamine were dramatically influenced in fed batch cultures resulting in major metabolic changes. Glucose limitation decreased lactate formation, but increased glutamine consumption and ammonium formation. Glutamine limitation decreased ammonium and alanine formation of lactate, alanine, and ammonium was negligible in the dual-substrate limited fed batch culture. The efficiency of the energy metabolism increased, as judged by the increase in the cellular yield coefficient for glucose by 100% and for glutamine by 150% and by the change in the metabolic ratios lac/glc, ala/ln, and NH(x)/ln, in the combined fed culture. The data indicate that a larger proportion of consumed glutamine enters the TCA cycle through the glutamate dehydrogenase pathway, which releases more energy from glutamine than the transamination pathway. We suggest that the main reasons for these changes are decreased uptake rates of glucose and glutamine, which in turn lead to a reduction of the pyruvate pool and a restriction of the flux through glutaminase and lactate dehydrogenase. There appears to be potential for further cell growth in the dual-substrate-limited fed batch culture as judged by a comparison of mu in the different cultures. (c) 1994 John Wiley & Sons, Inc.     

Metabolic rates of vascular endothelial cellsin vitro

Cultures of endothelial cells and cell lines of endothelial origin were maintained at confluence without medium exchange for a period of 72 h. During this time period the concentration of nutrients — amino acids and glucose — and metabolic waste products — lactate and ammonium — was determined as well as cell vitality and cell numbers. Metabolic rates were calculated and compared for the different cell lines. Surprisingly the primary cells showed significantly higher rates of glucose and glutamine consumption, respectively lactate production than the immortalized cell lines. Except for one tumorigenic cell line all cells showed a significant participation of transaminases in glutamine/ammonium metabolism. Furthermore it could be shown that in routine culture there was no depletion of nutrients or critical accumulation of ammonium or lactate over a culture period of 72 h.Abbreviations BAEC bovine aorta endothelial cells - EC vascular endothelial cells - FGF fibroblast growth factor - HUVEC vascular endothelial cells from human umbilical cord veins - IF 1:1 mixture of Iscove's MDM and Ham's F12 basal media - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid - NCS newborn calf serum - PBS phosphate buffered saline - TE 0.05% (w/V) trypsin, 0.02% (w/v) EDTA in PBS     

Kinetics and metabolic specificities of Vero cells in bioreactor cultures with serum-free medium

The aim of this study was to understand the metabolism kinetics of Vero cells grown on microcarriers in bioreactors in serum-free medium (SFM). We sought to determine what nutrients are essential for Vero cells and how they are consumed. Contrary to glucose and to most of the amino acids, glutamine and serine were very quickly depleted in this medium and can be supposed to be responsible for cell apoptosis. Lactate and ammonium ions did not reach toxic levels for Vero cells. We payed more attention to the lactate metabolism. Usually we observed that after about 2 days lactate was consumed in serum-containing media, but its concentration plateaud in SFM. Moreover, the addition of serum in SFM provoked lactate consumption and the rate of glucose and glutamine consumption was twice as high as in the SFM not supplemented with serum. The depletion of glutamine and serine and the metabolic deviations leading to a shortage of intermediate products required for other metabolic pathways probably contribute to the lower cell yield and higher cell death rate in SFM. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transient responses of hybridoma metabolism to changes in the oxygen supply rate in continuous culture

Oxygen is an important nutrient that may limit the productivity of commercial cell culture reactors. The transient responses of hybridoma growth and metabolism to step changes in the oxygen supply rate have been examined for dissolved oxygen concentrations (DO) ranging from 0.1% to 10% of air saturation in continuous culture. Metabolic quotients are reported for glucose, lactate, ammonia, oxygen, glutamine, alanine and other amino acids. A majority of the estimated ATP production was due to oxidative phosphorylation under all conditions tested. Decreases in the oxygen supply rate below the value required to maintain 0.5% DO caused the viable cell concentration to decrease. Glycolysis was enhanced at the lower oxygen concentrations, and after an initial decrease, the specific glutamine consumption rate was also higher. High residual glutamine concentrations occurred below 0.5% DO. Oxidation of other amino acids and production of serine were also inhibited. The cells subsequently adapted to low oxygen concentrations. The increase in cell concentration following the return to 10% DO was preceded by increased biosynthetic activity, as evidenced by transiently reduced yields of lactate from glucose, and alanine and ammonia from glutamine.     

Kinetic analysis of hybridoma cell culture in a protein-free medium: Substrate and agitation effects

A kinetic study of a hybridoma cell line that produces monoclonal antibodies against lactoferrin was carried out. A well defined protein-free culture medium was employed to facilitate the subsequent purification of the monoclonal antibodies. It should be highlighted that most of the existing work has been carried out employing culture media enriched with fetal bovine serum (FBS). Cell growth and monoclonal antibody production were monitored and kinetic parameters were determined. Besides, fundamental nutrients such as glucose and glutamine, inhibitory products such as ammonium and lactate, and several amino acids were followed throughout the culture. Additional experiments were carried out supplementing the medium with glutamine and ammonium, none of them resulting the key compound that halted the cell growth under the tested conditions and an unstructured model can be used to describe the system. Finally, agitation of the culture by a rocker set-up has shown high values of the specific death rate.     

The transient responses of hybridoma cells to nutrient additions in continuous culture: II. Glutamine pulse and step changes

The transient and steady-state responses of hybridoma growth and metabolism to glutamine pulse and step changes have been examined. Metabolic quotients are reported for oxygen, glucose, lactate, ammonia, glutamine, alanine, and other amino acids. The specific glutamine consumption rate increased rapidly after all glutamine additions, but the responses of the glucose and oxygen consumption rates and the cell concentration were found to depend on the intial feed glutamine concentration. The glucose consumption rate was 1.4-10.9 times that of glutamine, and serine and branched-chain amino acids were consumed in larger amounts at the higher glucose: glutamine uptake ratios. It was estimated that maintenance accounted for ca. 60% of the cellular ATP requirements at specific growth rates ranging from 0.57 to 0.68 day(-1).     

Alteration of mammalian cell metabolism by dynamic nutrient feeding

The metabolism of hybridoma cells was controlled to reduce metabolic formation in fed-batch cultures by dynamically feeding a salt-free nutrient concentrate. For this purpose, on-line oxygen uptake rate (OUR) measurement was used to estimate the metabolic demand of hybridoma cells and to determine the feeding rate of a concentrated solution of salt-free DMEM/F12 medium supplemented with other medium components. The ratios among glucose, glutamine and other medium components in the feeding nutrient concentrate were adjusted stoichiometrically to provide balanced nutrient conditions for cell growth. Through on-line control of the feeding rate of the nutrient concentrate, both glucose and glutamine concentrations were maintained at low levels of 0.5 and 0.2 mM respectively during the growth stage. The concentrations of the other essential amino acids were also maintained without large fluctuations. The cell metabolism was altered from that observed in batch cultures resulting in a significant reduction of lactate, ammonia and alanine production. Compared to a previously reported fed-batch culture in which only glucose was maintained at a low level and only a reduced lactate production was observed, this culture has also reduced the production of other metabolites, such as ammonium and alanine. As a result, a high viable cell concentration of more than 1.0 × 10⁷ cells/mL was achieved and sustained over an extended period. The results demonstrate an efficient nutrient feeding strategy for controlling cell metabolism to achieve and sustain a high viable cell concentration in fed-batch mammalian cell cultures in order to enhance the productivity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nutrient enrichment and in-situ waste removal through electrical means for hybridoma cultures

In-situ dc electric fields were applied to remove ammonium and lactate from suspension hybridoma cultures (ATCC-CRL-1606) which used enriched media. Nutrient concentration was increased fourfold above the normal concentration of DMEM to study enhanced protein product formation in a dc electric field. In the presence of the electric field, hybridoma growth and antibody production were increased 1.5-fold (from 3.7 x 10(6) to 9.1 x 10(6) viable cells/mL) and twofold (from 170 to 505 mg IgG/L), respectively, compared with the control. The effective removal of ammonium and lactate and increased concentrations of the various nutrients accounted for this enhancement. The enriched media caused the overflow metabolism of glucose, glutamine, and various essential amino acids. The overconsumption of glucose also produced substantial amounts of lactate, which in turn greatly increased the medium osmolarity. The increase in medium osmolarity is believed to be one of the causes of cell death in these culture systems.(c) 1995 John Wiley & Sons, Inc.     

Transient responses of hybridoma cells to nutrient additions in continuous culture: I. Glucose pulse and step changes

Glucose and glutamine are the main nutrients used by mammalian cells in culture. Each provides unique biosynthetic precursors but are complementary for production of other metabolites and energy. The transient and steady-state responses of hybridoma growth and metabolism to glucose pulse and step changes have been examined. Metabolic quotients are reported for oxygen, glucose, lactate, ammonia, glutamine, alanine, and other amino acids. The glucose consumption rate increased by 100-200% immediately after glucose was added to the reactor, and the increased glycolytic ATP production appears to be responsible for the concurrent rapid decrease in the oxygen consumption rate. The effects on glutamine consumption were delayed, probably due to buffering by the TCA cycle and interrelated pathways. A period of increased biosynthetic activity, as evidenced by an increase in the estimated specific ATP production rate and lower by-product yields from glutamine, preceded the increase in cell concentration after the glucose step change. The biosynthetic yield of cells from ATP was calculated, and it was estimated that maintenance accounted for about 60% of the energy used by the cells at a specific growth rate of 0.66 day(-1). The estimated 22% ATP production due to glycoysis was twice as great as that before the step change.     

Chemometric evaluation of on-line high-pressure liquid chromatography in mammalian cell cultures: analysis of amino acids and glucose.

An on-line high-pressure liquid chromatography (HPLC) system capable of measuring amino acids and carbohydrates was used to study metabolism in mammalian cell culture systems. The HPLC method utilized anion-exchange chromatography followed by integrated pulsed amperometric detection. The method is capable of measuring 19 amino acids plus glucose with a complete method time of 65 min. In actual cell cultures, the method was shown to be useful for monitoring 17 amino acids plus glucose. The two amino acids that were not accurately monitored were arginine and lysine, possibly due to their elution near the void volume of the column. The HPLC system was used to study variability in metabolism across different cell culture processes, as well as the effect of glucose and glutamine limitation on a single cell culture process. Chemometric analysis was used to draw statistically meaningful conclusions from the highly correlated, multivariate data set that resulted from these experiments. Using chemometrics, variation between processes was linked to differences in uptake rates of seven amino acids. Similarly, lactate concentration, cell density, and aspartate uptake rate were linked to glucose and glutamine limitation. The effect of nutrient limitation on glutamate, alanine, and ammonium was also considered.     

Growth and metabolism of marine fish Chinook salmon embryo cells: response to lack of glucose and glutamine

A peculiar phenomenon, differing from the response of mammalian cells, occurred when Chinook salmon embryo (CHSE) cells were passaged in the medium lacking of both glucose and glutamine. To elucidate metabolic mechanism of CHSE cells, the metabolism parameters, key metabolic enzymes, and ATP levels were measured at different glucose and glutamine concentrations. In the glutamine-free culture, hexokinase activity kept constant, and lactate dehydrogenase (LDH) activity decreased. This indicated that lack of glutamine did not expedite glucose consumption but made it shift to lower lactate production and more efficient energy metabolism. The results coincided with the experimental results of unaltered specific glucose consumption rate and decreased yield coefficients of lactate to glucose. In the glucose-free culture, simultaneous increase of glutaminase activity and of specific ammonia production rate suggested an increased flux into the glutaminolysis pathway, and increases of both glutamate dehydrogenase activity and yield coefficient of ammonia to glutamine showed an increased flux into deamination pathway. However, when glucose and glutamine were both lacking, the specific consumption rates of most of amino acids increased markedly, together with decrease of LDH activity, indicating that pyruvate derived from amino acids, away from lactate production, remedied energy deficiency. When both glucose and glutamine were absent, intracellular ATP contents and the energy charge remained virtually unaltered.Revisions requested 16 December 2004; Revisions received 24 January 2005     

重组CHO细胞培养过程中氨对细胞代谢的影响

研究了重组CHO细胞批培养过程中,氨浓度对细胞的葡萄糖、谷氨酰胺及其它氨基酸代谢的影响。表明,细胞对葡萄糖和谷氨酰胺的得率系数随着氨浓度的增加而降低,起始氨浓度为566mmol/L的批培养过程与起始氨浓度为021mmol/L的批培养过程相比,细胞对葡萄糖和谷氨酰胺的得率系数分别下降了78%和74%,细胞对其它氨基酸的得率系数也分别下降了50%～70%。氨浓度的增加明显地改变了细胞的代谢途径,葡萄糖代谢更倾向于厌氧的乳酸生成。在谷氨酰胺的代谢过程中,谷氨酸经谷氨酸脱氢酶进一步生成α酮戊二酸的过程受到了氨的抑制,而氨对谷氨酸经谷氨酸转氨酶反应生成α酮戊二酸的过程有促进作用,但总体上谷氨酸进一步脱氨生成α酮戊二酸的反应受到了氨的限制。     

Adaptive control at low glucose concentration of HEK-293 cell serum-free cultures.

Fed-batch cultures were implemented to study the metabolism of HEK-293 cells. Glucose, measured every 30 min by a FIA biosensor system, was maintained at 1 mM throughout the culture using an adaptive nonlinear controller based on minimal process modeling. The controller performed satisfactorily at both low and high cell concentrations without the need for retuning between different culture phases. Overall, lactate production was significantly reduced by maintaining a low glucose concentration, thus decreasing the rate of glycolysis. The rates of glucose and glutamine uptake as well as the lactate and ammonia production were compared to those obtained in batch mode with an initial glucose concentration of 21 mM. Basically, three phases were observed in both culture modes. The metabolic shift from the first to the second phase was characterized by a significant reduction in glucose consumption and lactate production while maximum growth rate was maintained. The specific respiration rate appeared unchanged during the first two phases, suggesting that no change occurred in the oxidative pathway capacity. In the third phase, cell growth became slower very likely due to glutamine limitation.