Abstract
Quantitative estimates of intracellular fluxes and measurements of intracellular concentrations were used to evaluate the effect of dissolved oxygen (DO) concentration on CRL 1606 hybridoma cells in batch culture. The estimates of intracellular fluxes were generated by combining material balances with measurements of extracellular metabolite rates of change. Experiments were performed at DO levels of 60% and 1% air saturation, as well as under oxygen-limited conditions. Cell extracts were analyzed to evaluate the effect of DO on the intracellular concentrations of the glutamate dehydrogenase reactants, as well as the redox state of the pyridine nucleotides in the cytosol and mitochondria. The relationship between cell density and pyridine nucleotide redox state was also investigated. Dissolved oxygen concentration had a significant effect on nitrogen metabolism and the flux through glutamate dehydrogenase was found to reverse at low DO, favoring glutamate formation. The NAD in the cytosol and mitochondria was more reduced under low DO conditions while the cytosolic NAD was more oxidized at low DO. Cytosolic NAD was reduced at higher cell densities while the redox states of cytosolic NADP and mitochondrial NAD did not exhibit significant variation with cell density. These results point to the fundamental role of the intracellular oxidation/reduction state in cell physiology and the possibility of controlling physiological processes through modulation of the dissolved oxygen level or the oxidation/reduction potential of the culture.
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References
Boraston R, Thompson PW, Garland S, Birch JR (1984) Growth and oxygen requirements of antibody producing mouse hybridoma cells in suspension culture. Dev Biol Stand 55:103–111
Bücher T, Sies H (1976) Mitochondrial and cytosolic redox states in perfused rat liver:methods and problems in metabolic compartmentation. In:Tager JM, Söling HD, Williamson JR (eds.) Use of isolated liver cells and kidney tubules in metabolic studies. North-Holland, Amsterdam, pp 41–64
Crane FL, Sun IL, Clark MG, Grebing C, Löw H (1985) Transplasma-membrane redox systems in growth and development. Biochim Biophys Acta 811:233–264
Fleischaker RJ (1982) An experimental study in the use of instrumentation to analyze metabolism and product formation in cell culture. PhD thesis, Massachusetts Institute of Technology
Hoek JB, Rydström J (1988) Physiological roles of nicotinamide nucleotide transhydrogenase. Biochem J 254:1–10
Hwang C (1992) The analysis of intracellular and extracellular redox states in cell culture. PhD thesis, Biochemical Engineering, Massachusetts Institute of Technology
Hwang C, Sinskey AJ (1991) The role of oxidation reduction potential in monitoring growth of cultured mammalian cells. In: Spier RE, Griffiths JB, Meigner B (eds) Production of biologicals from animal cells in culture. Halley Court, Oxford, pp 548–567
Hwang C, Sinskey AJ, Lodish HF (1992) Oxidized redox state of glutathione in the endoplasmic reticulum. Science 257:1496–1502
Kilburn DG, Lilly MD, Self DA, Webb FC (1969) The effect of dissolved oxygen partial pressure on the growth and carbohydrate metabolism of mouse LS cells. J Cell Sci 4:25–37
Klepper O, Van de Kamer JPG (1987) The use of mass balances to test and improve the estimates of carbon fluxes in an ecosystem. Math Biosci 85:37–49
Kosower NS, Kosower EM (1978) The glutathione status of cells. Int Rev Cytol 54:109–160
Krebs HA, Veech RL (1969) Pyridine nucleotide interrelations. In: Papa S, Tager JM, Quagliariello E, Slater EC (eds) The energy level and metabolic control in mitochondria. Adriatica, Bari, pp 329–382
Lowry OH, Passonneau JV (1972) A flexible system of enzymatic analysis. Academic Press, New York
Meersman RED, Faroudja NY (1988) Computerized substrate utilization determinations from respiratory functions alone. Comput Biol Med 18:449–453
Meister A, Anderson ME (1983) Glutathione. Annu Rev Biochem 52:711–760
Miller WM, Wilke CR, Blanch HW (1987) Effects of dissolved oxygen concentration on hybridoma growth and metabolism in continuous culture. J Cell Physiol 132:524–530
Miller WM, Wilke CR, Blanch HW (1988) Transient responses of hybridoma metabolism to changes in the oxygen supply rate in continuous culture. Bioprocess Eng 3:113
Mizrahi A (1984) Oxygen in human lymphoblastoid cell line cultures and effect of polymers in agitated and aerated cultures. Dev Biol Stand 55:93–102
O'Donnel-Tormey J, Nathan CF, Lanks K, DedBoer CJ, Harpe J de la (1987) Secretion of pyruvate:an antioxidant defense of mammalian cells. J Exp Med 165:500–514
Ozturk SS, Palsson BØ (1990a) Chemical decomposition of glutamine in cell culture media:effect of media type, pH, and serum concentration. Biotechnol Prog 6:121–128
Ozturk SS, Palsson BØ (1990b) Effects of dissolved oxygen on hybridoma cell growth, metabolism, and antibody production kinetics in continuous culture. Biotechnol Prog 6:437–446
Phillips HA, Scharer JM, Bols NC, Moo-Young M (1987) Effect of oxygen on antibody productivity in hybridoma culture. Biotechnol Lett 9:745–750
Reardon KF, Scheper T-H, Bailery JE (1987) Metabolic pathway rates and culture fluorescence in batch fermentations of Clostridium acetobutylicum. Biotechnol Prog 3:153–167
Reuveny S, Velez D, Macmillan JD, Miller L (1986) Factors affecting cell growth and monoclonal antibody production in stirred reactors. J Immunol Methods 86:53–59
Rydström J, Persson B, Carlenor E (1987) Transhydrogenases linked to pyridine nucleotides. In:Dolphin D, Avromovic O, Poulson R (eds) Pyridine nucleotide coenzymes. Wiley, New York, pp 433–460
Safer B, Williamson JR (1973) Mitochondrial-cytosolic interactions in perfused rat heart. J Biol Chem 248:2570–2579
Siano SA, Mutharasan R (1991) NADH fluorescence and oxygen uptake responses of hybridoma cultures to substrate pulse and step changes. Biotechnol Bioeng 37:141–159
Tischler ME, Friedrichs D, Coll K, Williamson JR (1977) Pyridine nucleotide distributions and enzyme mass action ratios in hepatocytes from fed and starved rats. Arch Biochem Biophys 183:222–236
Vallino JJ, Stephanopoulos G (1989) Flux determination in cellular bioreaction networks:applications to lysine fermentations. In: Sikdar SK, Bier M, Todd P (eds) Frontiers in bioprocessing. CRC, Boca Raton, Fla, pp 05–219
Veech RL (1987) Pyridine nucleotides and control of metabolic processes. In: Dolphin D, Avromovic O, Poulson R (eds) Pyridine nucleotide coenzymes. Wiley, New York, pp 79–104
Verhoff FH, Spradlin JE (1976) Mass and energy balance analysis of metabolic pathways applied to citric acid production by Asperigillus niger. Biotechnol Bioeng 18:425–432
Wanders RJA, Meijer AJ, Groen AK, Tager JM (1983) Bicarbonate and the pathway of gluatamate oxidation in isolated rat-liver mitochondria. Eur J Biochem 133:245–254
Zupke C (1993) Metabolic flux analysis in mammalian cell culture. PhD thesis, Chemical Engineering, Massachusetts Institute of Technology
Zupke C, Stephanopoulos G (1995) Intracellular flux analysis in hybridomas using mass balances and in vitro 13C NMR. Biotechnol Bioeng 45:292–303
Zuurendonk PF, Akerboom TPM, Tager JM (1976) Metabolite distribution in isolated rat-liver cells and equilibrium relationships of mitochondrial and cytosolic dehydrogenases. In: Tager JM, Söling HD, Williamson JR (eds). Use of isolated liver cells and kidney tubules in metabolic studies. North-Holland, Amsterdam, pp 17–27
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Zupke, C., Sinskey, A.J. & Stephanopoulos, G. Intracellular flux analysis applied to the effect of dissolved oxygen on hybridomas. Appl Microbiol Biotechnol 44, 27–36 (1995). https://doi.org/10.1007/BF00164476
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DOI: https://doi.org/10.1007/BF00164476