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. 2003 May;42(1):1–11. doi: 10.1023/A:1026185615650

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

Sébastien Quesney 1, Annie Marc 2, Catherine Gerdil 3, Cyrille Gimenez 3, Jacqueline Marvel 4, Yves Richard 5, Bernard Meignier 3
PMCID: PMC3449507  PMID: 19002923

Abstract

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.

Keywords: Amino acid, Glucose, Lactate, Metabolism, Microcarriers, Serum-free medium, Vero cells

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References

  1. Abercrombie M., Ambrose E.J. The surface properties of cancer cells: A review. Cancer Res. 1962;22:525–548. [PubMed] [Google Scholar]
  2. Al-Rubeai M. Apoptosis and cell culture technology. Adv Biochem Eng Biotechnol. 1998;59:225–249. doi: 10.1007/BFb0102300. [DOI] [PubMed] [Google Scholar]
  3. Bonarius H.P.J., Hatzimanikatis V., Meesters K.P.H., de Gooijer C.D., Schmid G., Tramper J. Metabolic flux analysis of hybridoma cells in different culture media using mass balances. Biotechnol Bioeng. 1995;50:299–318. doi: 10.1002/(SICI)1097-0290(19960505)50:3<299::AID-BIT9>3.0.CO;2-B. [DOI] [PubMed] [Google Scholar]
  4. Clark J.M., Gebb C., Hirtenstein M.D. Serum supplements and serum-free media: applicability for microcarrier culture of animal cells. Devel Biol Stand. 1982;50:81–91. [PubMed] [Google Scholar]
  5. Cruz J.H., Moreira J.L., Carrondo M.J.T. Metabolic shifts by nutrient manipulation in continuous cultures of BHK cells. Biotechnol Bioeng. 1999;66:104–113. doi: 10.1002/(SICI)1097-0290(1999)66:2<104::AID-BIT3>3.0.CO;2-#. [DOI] [PubMed] [Google Scholar]
  6. Doverskog M., Ljunggren J., Öhaman L., Häggström L. Physiology of cultured animal cells. J BioTechnol. 1992;59:103–115. doi: 10.1016/S0168-1656(97)00172-7. [DOI] [PubMed] [Google Scholar]
  7. Duval D., Demangel C., Miossec S., Geahel I. Role of metabolic waste products in the control of cell proliferation and antibody production by mouse hybridoma cells. Hybridoma. 1992;11:311–322. doi: 10.1089/hyb.1992.11.311. [DOI] [PubMed] [Google Scholar]
  8. Europa A.F., Gambhir A., Fu P.G., Hu W.S. Multiple steady states with distinct metabolism in continuous culture of mammalian cells. Biotechnol Bioeng. 1999;67:25–34. doi: 10.1002/(SICI)1097-0290(20000105)67:1<25::AID-BIT4>3.0.CO;2-K. [DOI] [PubMed] [Google Scholar]
  9. Fenge C., Klein C., Heuer C., Siegel U., Fraune E. Agitation, aeration and perfusion modules for cell culture bioreactors. Cytotechnology. 1993;11:233–244. doi: 10.1007/BF00749874. [DOI] [PubMed] [Google Scholar]
  10. Glacken M.W. Catabolic control of mammalian cell culture. Biotechnol. 1988;6:1041–1050. doi: 10.1038/nbt0988-1041. [DOI] [Google Scholar]
  11. Gstraunthaler G., Seppi T., Pfaller W. Impact of culture conditions, culture media volume, and glucose content on metabolic properties of renal epithelial cell cultures. Cell Physiol Biochem. 1999;9:150–172. doi: 10.1159/000016312. [DOI] [PubMed] [Google Scholar]
  12. Guppy M., Greiner E., Brand K. The role of the crabtree effect and endogenous fuel in the energy metabolism of resting and proliferating thymocytes. Eur J Biochem. 1993;212:95–99. doi: 10.1111/j.1432-1033.1993.tb17637.x. [DOI] [PubMed] [Google Scholar]
  13. Hassel T., Gleave S., Butler M. Growth inhibition in animal cell culture. The effect of lactate and ammonia. Appl Biochem Biotechno. 1990;30:29–41. doi: 10.1007/BF02922022. [DOI] [PubMed] [Google Scholar]
  14. Irani N., Wirth M., Van Den Heuvel J., Wagner R. Improvement of the primary metabolism of cell cultures by introducing a new cytoplasmic pyruvate carboxylase reaction. Biotechnol Bioeng. 1999;66:238–246. doi: 10.1002/(SICI)1097-0290(1999)66:4<238::AID-BIT5>3.0.CO;2-6. [DOI] [PubMed] [Google Scholar]
  15. Lee G.T-Y., Engelhardt D.L. Protein metabolism during growth of Vero cells. J Cell Physiol. 1977;92:293–302. doi: 10.1002/jcp.1040920218. [DOI] [PubMed] [Google Scholar]
  16. Linz M., Zeng A.P., Wagner R., Deckwer W.D. Stoichiometry, kinetics and regulation of glucose and aminoacid metabolism of a recombinant BHK cell line in batch and continuous cultures. Biotechnol Prog. 1997;13:453–463. doi: 10.1021/bp970032z. [DOI] [PubMed] [Google Scholar]
  17. Mendonça R.Z., Ioshimoto L.M., Mendonça R.M.Z., De-Franco M., Valentini E.J.G., Beçak W., et al. Preparation of human rabies vaccine in Vero cell culture using a microcarrier system. Braz J Med Biol Res. 1993;26:1305–1317. [PubMed] [Google Scholar]
  18. Mendonça R.Z., Pereira C.A. Cell metabolism and medium perfusion in Vero cell cultures on microcarriers in a bioreactor. Bioprocess Eng. 1997;18:213–218. doi: 10.1007/s004490050433. [DOI] [Google Scholar]
  19. Merten O.W., Kierulff J.V., Castignolles N., Perrin P. Evaluation of the new serum-free medium (MDSS2) for the production of different biologicals: Use of various cell lines. Cytotechnology. 1994;14:47–59. doi: 10.1007/BF00772195. [DOI] [PubMed] [Google Scholar]
  20. Montagnon J.B., Vincent-Falquet J.C., Fanget B. Thousand litre scale microcarrier culture of Vero cells for killed polio virus vaccine. Promising results. Devel Biol Stand. 1984;55:37–42. [PubMed] [Google Scholar]
  21. Montagnon J.B., Fanget B., Vincent-Falquet J.C. Industrial-scale production of inactivated poliovirus vaccine prepared by culture of Vero cells on microcarrier. Rev Infect Dis. 1984;6:341–344. doi: 10.1093/clinids/6.supplement_2.s341. [DOI] [PubMed] [Google Scholar]
  22. Nahapetian A.T., Thomas J.N., Thilly W.G. Optimization of environment for high density Vero cell culture: effect of dissolved oxygen and nutrient supply on cell growth and changes in metabolites. J Cell Sci. 1986;81:65–103. doi: 10.1242/jcs.81.1.65. [DOI] [PubMed] [Google Scholar]
  23. Newsholme E.A., Crabtree B., Ardawi M.S.M. The role of high rates of glycolysis and glutamine utilization in rapidly dividing cells. Bioscience Rep. 1985;5:393–400. doi: 10.1007/BF01116556. [DOI] [PubMed] [Google Scholar]
  24. Nyberg G.B., Balcarcel R.R., Follstad B.D., Stephanopoulos G. and Wang D.I.C. 1999. Metabolism of peptide amnoacids by Chinese Hamster Ovary cells grown in a complex medium. (tiBiotechnol Bioeng 62: 324–335. [PubMed]
  25. Oller A.R., Buser C.W., Tyo M.A., Thilly W.G. Growth of mammalian cells at high oxygen concentrations. J Cell Sci. 1989;94:43–49. doi: 10.1242/jcs.94.1.43. [DOI] [PubMed] [Google Scholar]
  26. Ozturk S.S., Jorjani P., Taticek R., Lowe B., Shackleford S., Ladehoff-Guiles D., et al. et al. Kinetics of glucose metabolism and utilization of lactate in mammalian cell cultures. In: Carrondo M.J.T., et al.et al., editors. Animal Cell Technology. Netherlands: Kluwer Academic Publishers; 1997. pp. 355–360. [Google Scholar]
  27. Papaconstantinou H.T., Hwang K.O., Rajaraman S., Hellmich M.R., Townsend C.M., Ko T.C. Glutamine deprivation induces apoptosis in intestinal epithelial cells. Surgery. 1998;124:152–160. [PubMed] [Google Scholar]
  28. Pardridge W.M., Casanello-Ertl D. Effects of glutamine deprivation on glucose and amino acid metabolism in tissue culture. Am J Physiol. 1979;236:234–238. doi: 10.1152/ajpendo.1979.236.3.E234. [DOI] [PubMed] [Google Scholar]
  29. Philips H.J. In: Tissue cultures: Methods and Applications. Kruse P.F., Patterson M.K., editors. New York: Academic Press; 1973. pp. 406–408. [Google Scholar]
  30. Polastri G.D., Friensen H.J., Mauler R. Aminoacid utilisation by Vero cells in microcarrier culture. Devel Biol Stand. 1982;55:53–56. [PubMed] [Google Scholar]
  31. Quesney S., Marvel J., Marc A., Gerdil C., Meignier B. Characterization of Vero cell growth and death in bioreactor with serum-containing and serum-free media. Cytotechnology. 2001;35:115–125. doi: 10.1023/A:1017589526145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sakagami H., Satoh M., Yokote Y., Takano H., Takahama M., Kochi M., et al. Amino acid utilization during cell growth and apoptosis induction. Anticancer Res. 1998;18:4303–4306. [PubMed] [Google Scholar]
  33. Sandford K.K., Earle W.R., Evans V.J., Waltz H., Shannon J.E. The measurement of proliferation in tissue cultures by enumeration of cell nuclei. J Natl Cancer Inst. 1951;11:773–795. [PubMed] [Google Scholar]
  34. Sanfeliu A., Paredes C., Cairo J.J., Godia F., et al. Analysis of glucose and glutamine metabolism of hybridoma cells by continuous culture experiments. In: Carrondo M.J.T., et al., editors. Animal Cell Technology. Netherlands: Kluwer Academic Publishers; 1997. pp. 785–789. [Google Scholar]
  35. Van Wezel A.L. Microcarrier cultures of animals cells. In: Kruse P.F., Patterson M.K., editors. Tissue cultures: Methods and Applications. New York: Academic Press; 1973. pp. 372–377. [Google Scholar]
  36. Wagner A., Marc A., Engasser J.M., Einsele A. Growth and metabolism of human tumor kidney cells on galactose and glucose. Cytotechnology. 1991;7:7–13. doi: 10.1007/BF00135633. [DOI] [PubMed] [Google Scholar]
  37. Yasumura Y., Kawakita Y. Studies on SV40 in tissue culture – Preliminary step for cancer research in vitro. Nihon Rinsho (in Japanese) 1963;21:1201–1221. [Google Scholar]
  38. Zielke H.R., Zielke C.L., Ozand P.T. Glutamine: a major energy source for cultured mammalian cells. Fed Proc. 1984;43:121–125. [PubMed] [Google Scholar]
  39. Zielke H.R., Sumbilla C.M., Sevdalain D.A. Lactate: a major product of glutamine metabolism by human diploid fibroblasts. J Cell Physiol. 1980;104:433–441. doi: 10.1002/jcp.1041040316. [DOI] [PubMed] [Google Scholar]

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