Applied Microbiology and Biotechnology

, Volume 36, Issue 5, pp 621–625 | Cite as

Extra- and intracellular metabolite concentrations for murine hybridoma cells

  • Georg Schmid
  • Harvey W. Blanch
Biotechnology Short Contribution

Summary

Intra- and extracellular metabolite concentrations were determined for hybridoma cells grown in tissue culture flasks in batch culture. Significant differences were found for intracellular lactate and amino acid concentrations depending on the culture medium. AB2-143.2 cells cultivated in Dulbecco's modified Eagle's (DME) medium supplemented with 50 mm lactate exhibited intracellular lactate and glutamine levels of 40 mm and 4 mm, respectively, whereas cells grown in standard DME medium had low intracellular glutamine and 20 mm lactate concentrations. For cells cultivated in medium supplemented with 6 mm pyruvate, intracellular lactate levels were estimated at approx. 40 mm, but these cells also showed an increased intracellular alanine concentration of 22 mm. The higher alanine pools probably result from increased transamination of pyruvate under these conditions.

Keywords

Pyruvate Glutamine Metabolite Concentration Amino Acid Concentration Tissue Culture Flask 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bergström J, Fürst P, Noree L-O, Vinnars E (1974) Intracellular free amino acid concentration in human muscle tissue. J Appl Physiol 36:693–697Google Scholar
  2. Butler J DeB, Spielberg SP (1981) Decrease of intracellular cystine content in cystinotic fibroblasts by inhibitors of γ-glutamyl transpeptidase. J Biol Chem 256:4160–4163Google Scholar
  3. Clark RM, Chandler PT, Park CS, Norman AW (1980) Extracellular amino acid effects on milk protein synthesis and intracellular amino acid pools with bovine mammary cells in culture. J Dairy Sci 63:1230–1234Google Scholar
  4. Darmaun D, Matthews DE, Desjeux J-F, Bier DM (1988) Glutamine and glutamate nitrogen exchangeable pools in cultured fibroblasts: a stable isotope study. J Cell Physiol 134:143–148Google Scholar
  5. Delhotal B, Lemonnier F, Couturier M, Lemonnier A (1983) Influence of two cell harvesting methods on intracellular ATP and amino acid concentrations in human fibroblast cultures. Biochimie 65:121–125Google Scholar
  6. Eagle H (1959) Amino acid metabolism in mammalian cell culture. Science 130:432–437Google Scholar
  7. Eagle H, Piez KA, Levy M (1961) The intracellular amino acid concentrations required for protein synthesis in cultured human cells. J Biol Chem 236:2039–2042Google Scholar
  8. Eigenbrodt E, Fister P, Reinacher M (1985), New perspectives on carbohydrate metabolism in tumor cells. In: Beitner R (ed) Regulation of carbohydrate metabolism, vol. II. CRC Press, Boca Raton, pp 141–179Google Scholar
  9. Fernandez EJ, Mancuso A, Clark DS (1988) NMR spectroscopy studies of hybridoma metabolism in a simple membrane reactor. Biotechnol Prog 4:173–183Google Scholar
  10. Fernandez EJ, Mancuso A, Murphy MK, Blanch HW, Clark DS (1990) Nuclear magnetic resonance methods for observing the intracellular environment of mammalian cells. Ann NY Acad Sci 589:458–475Google Scholar
  11. Goh CJ, Dumbroff EB, Lepock JR (1988) Amino acid pools in CHL V79 cells during induction of thermotolerance: reduction in free intracellular glutamine. J Cell Physiol 135:139–144Google Scholar
  12. Hod Y, Hershko A (1976) Relationship of the pool of intracellular valine to protein synthesis and degradation in cultured cells. J Biol Chem 251:4458–4467Google Scholar
  13. Holopainen I, Oja SS, Marnela K-M, Kontro P (1986) Free amino acids of rat astrocytes in primary culture: changes during cell maturation. Int J Dev Neurosci 4:493–496Google Scholar
  14. Hornbeck PV, Lewis GK (1985) Idiotype connectance in the immune system II. A heavy chain variable region idiotope that dominates the antibody response to the p-azobenzenearsonate group is a minor idiotope in the response to trinitrophenyl group. J Exp Med 161:53–61Google Scholar
  15. Jonas AJ, Smith SA, Gochman N, Schneider JA (1982) Measurement of intracellular amino acids in cultured fibroblasts. Clin Chim Acta 120:321–329Google Scholar
  16. Kuchler RJ (1963) The flexible nature of the amino acid pool in L strain fibroblasts. Proc Soc Exp Biol Med 116:20–25Google Scholar
  17. Melancon SB, Tayco J, Nadler HL (1972) The free amino acid pool of cultivated human skin fibroblasts. Proc Soc Exp Biol Med 141:391–395Google Scholar
  18. Miller WM (1987) A kinetic analysis of hybridoma growth and metabolism. Ph. D. Thesis, University of California, BerkeleyGoogle Scholar
  19. Mohri T (1967) Effects of cortisol and 19-nortestosterone on the free amino acid pools and amino acid uptake of cultured cells. Endocrinology 81:454–460Google Scholar
  20. Myara I, Charpentier C, Gautier M, Lemonnier A (1985) Cell density affects prolidase and prolinase activity and intracellular amino acid levels in cultured human cells. Clin Chim Acta 150:1–9Google Scholar
  21. Oxender DL, Lee M, Cecchini G (1977) Regulation of amino acid transport activity and growth rate of animal cells in culture. J Biol Chem 252:2680–2683Google Scholar
  22. Patel AJ, Hunt A (1985) Concentration of free amino acids in primary cultures of neurones and astrocytes. J Neurochem 44:1816–1821Google Scholar
  23. Petronini PG, Tramacere M, Mazzini A, Kay JE, Borghetti AF (1989) Control of protein synthesis by extracellular Na+ in cultured fibroblasts. J Cell Physiol 140:202–211Google Scholar
  24. Piez KA, Eagle H (1958) The free amino acid pool of cultured human cells. J Biol Chem 231:533–545Google Scholar
  25. Reitzer LJ, Wice BM, Kennell D (1979) Evidence that glutamine, not sugar, is the major energy source for cultured HeLa cells. J Biol Chem 254:2669–2676Google Scholar
  26. Renner ED, Plagemann PGW, Bernlohr RW (1972) Permeation of glucose by simple and facilitated diffusion by Novikoff rat hepatoma cells in suspension culture and its relationship to glucose metabolism. J Biol Chem 247:5765–5776Google Scholar
  27. Sambuy Y, Bittles AH (1982) The effects of in vitro ageing on the composition of the intracellular free amino acid pool of human diploid fibroblasts. Mech Ageing Dev 20:279–287Google Scholar
  28. Schmid G, Johannsen R (1990) Metabolic quotients for recombinant CHO and BHK cell lines producing human antithrombin III. Biotechnol Lett 12:317–322Google Scholar
  29. Schmid G, Wilke CR, Blanch HW (1987) Continuous suspension culture of hybridomas with cell retention. Presented at the 194th ACS National Meeting, New Orleans, LA, USA, paper no. 74Google Scholar
  30. Schmid G, Blanch HW, Wilke CR (1990a) Hybridoma growth, metabolism, and product formation in HEPES-buffered medium. I. Effect of passage number. Biotechnol Lett 12:627–632Google Scholar
  31. Schmid G, Blanch HW, Wilke CR (1990b) Hybridoma growth, metabolism, and product formation in HEPES-buffered medium. II. Effect of pH. Biotechnol Lett 12:633–638Google Scholar
  32. Schmid G, Wilke CR, Blanch HW (1991) Continuous hybridoma suspension cultures with and without cell retention: kinetics of growth, metabolism, and product formation. J Biotechnol, in pressGoogle Scholar
  33. Silvotti L, Petronini PG, Mazzini A, Piedimonte G, Borghetti AF (1991) Differential adaptive response to hyperosmolarity of 3T3 and transformed SV3T3 cells. Exp Cell Res 193:253–261Google Scholar
  34. Wagner R, Ryll T, Krafft H, Lehmann J (1988) Variation of amino acid concentrations in the medium of HU β-IFN and HU IL-2 producing cell lines. Cytotechnology 1:145–150Google Scholar
  35. Wheatley DN, Inglis MS (1983) Is the intracellular amino acid pool expandable? IRCS Med Sci 11:365–366Google Scholar
  36. Wheatley DN, Inglis MS, Malone PC (1986) The concept of the intracellular amino acid pool and its relevance in the regulation of protein metabolism, with particular reference to mammalian cells. In: Horecker BL, Stadtman ER (eds) Current topics in cellular regulation, vol 28. Academic Press, Orlando, pp 107–182Google Scholar
  37. Yudkoff M, Nissim I, Nissim I, Stern J, Pleasure D (1989) Effects of palmitate on astrocyte amino acid contents. Neurochem Res 14:367–370Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Georg Schmid
    • 1
    • 2
  • Harvey W. Blanch
    • 1
  1. 1.Department of Chemical EngineeringUniversity of CaliforniaBerkeleyUSA
  2. 2.Pharma Research — New TechnologiesPRTMBasleSwitzerland

Personalised recommendations