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Adaptation of mammalian cells to growth in serum-free media

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Abstract

A three-step protocol is described for adapting an anchorage-dependent, serum-dependent recombinant mammalian cell lineage to high density serum-free suspension culture. The objective is a cell lineage that is well-suited for the manufacture of a recombinant protein. The first step of the protocol generates an anchorage-independent cell lineage by culturing trypsin-treated cells in spinner flasks using serum-containing medium. The second step adapts the lineage to serum-free medium through a series of serum reduction steps in the presence of defined growth-promoting additives. The third step adapts the lineage to high-cell-density conditions by culturing the cells in a bioreactor in a manner that allows development of tolerance to growth-inhibiting substances released by the cells. Examples are presented for the use of this protocol for recombinant CHO cells.

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References

  1. Lambert, K. J. and Birch, J. R. (1985) Cell growth media, in Animal Cell Biotechnology. Volume I. (Spier, R. E. and Griffiths, J. B., eds.), Academic Press, pp. 85–112.

  2. Miller, W. M., Wilke, C. R., and Blanch, H. W. (1988) Transient responses of hybridoma cells to lactate and ammonia pulse and step changes in continuous culture. Bioprocess Eng. 3, 113–122.

    Article  CAS  Google Scholar 

  3. Matsumura, M., Shimoda, M., Arii, T., and Kataoka, H. (1991) Adaptation of hybridoma cells to higher ammonia concentration. Cytotechnology 7, 103–112.

    PubMed  CAS  Google Scholar 

  4. Schumpp, B. and Schlaeger, E.-J. (1992) Growth study of lactate and ammonia double-resistant clones of HL-60 cells. Cytotechnology 8, 39–44.

    Article  PubMed  CAS  Google Scholar 

  5. Inlow, D., Maiorella, B., and Shauger, A. E. (1992) Methods for adapting cells for increased product production through exposure to ammonia, US Patent 5,156,964.

  6. Adamson, S. R., Drapeau, D., Luan, Y.-T., and Miller, D. A. (1996) Adaptation of mammalian cell lines to high cell densities, US Statutory Invention Registration H1532.

  7. Hayter, P. M., Curling, M. A., Baines, A. T., Jenkins, N., Salmon, I., Strange, P. G., and Bull, A. T. (1991) Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture. Appl. Microbiol. Biotechnol. 34, 559–564.

    Article  PubMed  CAS  Google Scholar 

  8. Murata, M., Eto, Y., and Shibai, H. (1988) Largescale production of erythroid differentiation factor (EDF) by gene-engineered Chinese hamster ovary (CHO) cells in suspension culture. J. Ferment. Technol. 66(5), 501–507.

    Article  CAS  Google Scholar 

  9. Berg, D. T., McClure, D. B., and Grinnell, B. W. (1993) High-level expression of secreted proteins from cells adapted to serum-free suspension culture. BioTechniques 14(6), 972–978.

    PubMed  CAS  Google Scholar 

  10. Broad, D., Boraston, R., and Rhodes, M. (1991) Production of recombinant proteins in serum-free media. Cytotechnology 5, 57–55.

    Article  Google Scholar 

  11. Mather, J. P. (1990) Optimizing cell and culture environment for production of recombinant proteins. Methods Enzymol. 185, 567–577.

    Article  PubMed  CAS  Google Scholar 

  12. Keen, M. J. and Nicholas, T. R. (1995) Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line. Cytotechnology 17, 153–163.

    Article  CAS  Google Scholar 

  13. Perrin, P., Madbusudana, S., Gontier-Jallet, C., Petres, S., Tordo, N., and Merten, O.-W. (1995) An experimental rabies vaccine produced with a new BHK-21 susponsion cell culture process use of serum-free medium and perfusion-reactor system. Vaccine 13, 1244–1250.

    Article  PubMed  CAS  Google Scholar 

  14. Sinacore, M. S., Charlebois, T. C., Harrison, S., Brennan, S., Richards, T., Hamilton, M., Scott, S., Brodeur, S., Oakes, P., Leonard, M., Switzer, M., Anagnostopoulos, A., Foster, B., Harris, A., Jankowski, M., Bond, M., Martin, S., and Adamson, S. R. (1996) CHO DUKX cell lineages preadapted to growth in serum-free suspension culture enable rapid development of cell culture processes for the manufacture of recombinant proteins. Biotechnol. Bioeng. 52, 518–528.

    Article  CAS  PubMed  Google Scholar 

  15. Zang, M., Trautmann, H., Gandor, C., Messi, F., Asselbergs, F., Leist, C, Fietcher, A., and Reiser, J. (1995) Production of recombinant proteins in Chinese hamster ovary cells using protein-free cell culture medium. Biotechnology 13, 389–392.

    Article  PubMed  CAS  Google Scholar 

  16. Gandor, C., Leist, C., Feichter, A., and Asselbergs, F. (1995) Amplification and expression of recombinant genes in serum-independent Chinese hamster ovary cells. FEBS Lett. 377, 290–294.

    Article  PubMed  CAS  Google Scholar 

  17. Kawamoto, T., Sato, J. D., Le, A., McClure, D. B., and Sato, G. H. (1983) Development of a serum-free medium for growth of NS-1 mouse myeloma cells and its application to the isolation of NS-1 hybridomas. Anal. Biochem. 130, 445–453.

    Article  PubMed  CAS  Google Scholar 

  18. Kovar, J. and Franek, F. (1984) Serum-free medium for hybridoma and parental myeloma cell cultivation: A novel composition of growth-supporting substances. Immunol. Lett. 7, 339–345.

    Article  PubMed  CAS  Google Scholar 

  19. Miyaji, H., Mizukami, T., Hosoi, S., Sato, S., Fujiyoshi, N., and Itoh, S. (1990) Expression of human beta-interferon in Namalwa KJM-1 which was adapted to serum-free medium. Cytotechnology 3, 133–140.

    Article  PubMed  CAS  Google Scholar 

  20. Griffiths, J. B. and Racher, A. J. (1994) Cultural and physiological factors affecting expression of recombinant proteins. Cytotechnology 15, 3–9.

    Article  PubMed  CAS  Google Scholar 

  21. Assoian, R. K. (1977) Anchorage-dependent cell cycle progression. J. Cell. Biol. 136(1), 1–4.

    Article  Google Scholar 

  22. Rouslahti, E. and Reed, J. C. (1994) Anchorage dependence, integrins and apoptosis. Cell 77, 477,478.

    Article  Google Scholar 

  23. Jeso, B. D., Ulianich, L., Racioppi, L., D’Armiento, F., Feliciello, A., Pacifico, F., Consiglio, E., and Fromisano, S. (1995) Serum withdrawal induced apoptotic cell death in K1-Ras transformed but not normal differentiated thyroid cells. Biochem. Biophys. Res. Commun. 214, 819–824.

    Article  PubMed  Google Scholar 

  24. Curling, E. M., Hayter, P. M., Baines, A. J., Bull, A. T., Gull, K., Strange, P. G., and Jenkins, N. (1990) Recombinant human interferon-γ: differences in glycosylation and proteolytic processing lead to heterogeneity in batch culture. Biochem. J. 272, 333–337.

    PubMed  CAS  Google Scholar 

  25. Watson, E., Shoh, B., Leiderman, L., Hsu, Y.-R., Lu, H. S., and Lin, F.-K. (1994) Comparison of N-linked oligosaccharides of recombinant human tissue kallikrein produced by Chinese hamster ovary cells in microcarriers and in serum-free suspension culture. Biotechnol. Prog. 10, 39–44.

    Article  PubMed  CAS  Google Scholar 

  26. Chotigeat, W., Watanapokasin, Y., Mahler, S., and Gray, P. P. (1994) Role of environmental conditions on the expression levels, glycoform pattern and levels of sialytransferase for hFSH produced by recombinant CHO cells. Cytotechnology 15, 217–221.

    Article  PubMed  CAS  Google Scholar 

  27. Lifely, M. R., Hale, C., Boyce, S., Keen, M. J., and Phillips, J. (1995) Glycosylation and biological activity of CAMPATH-1H expressed in different cell lines and grown under different culture conditions. Glycobiology 5, 813–822.

    Article  PubMed  CAS  Google Scholar 

  28. Jenkins, N., Parekh, R. B., and James, D. C. (1996) Getting the glycosylation right: implications for the biotechnology industry. Nature Biotechnol. 14, 975–981.

    Article  CAS  Google Scholar 

  29. Gawlitzek, M., Valley, U., Nimtz, M., Wagner, R., and Conradt, H. S. (1995) Characterization of changes in the glycosylation pattern of recombinant proteins from BHK-21 cells due to different culture conditions. J. Biotechnol. 42, 117–131.

    Article  PubMed  CAS  Google Scholar 

  30. Ozturk, S. S. and Palsson, B. O. (1991) Physiological changes during the adaptation of hybridoma cells to low serum and serum-free media. Biotechnol. Bioeng. 37, 35–46.

    Article  CAS  PubMed  Google Scholar 

  31. Barnes, D. and Sato, G. (1980) Methods for growth of cultured cells in serum-free medium. Anal. Biochem. 102, 255–270.

    Article  PubMed  CAS  Google Scholar 

  32. Merten, Q.-W., Kierulff, J. V., Castignolles, N., and Perrin, P. (1994) Evaluation of the new serum-free medium (MDSS2) for the production of different biologicals: use of various cell lines. Cytotechnology 14, 47–59.

    Article  PubMed  CAS  Google Scholar 

  33. Radford, K., Niloperbowo, W., Reid, S., and Greenfield, P. F. (1991) Weaning of three hybridoma cell lines to serum free low protein medium. Cytotechnology 6, 65–78.

    Article  PubMed  CAS  Google Scholar 

  34. Qi, Y. M., Greenfield, P. F., and Reid, S. (1996) Evaluation of a simple protein free medium that supports high levels of monoclonal antibody production. Cytotechnology 21, 95–109.

    Article  CAS  Google Scholar 

  35. Chang, T. H., Steplewski, Z., and Koprowski, H. (1980) Production of monoclonal antibodies in serumfree medium. J. Immunol. Methods 39, 369–375.

    Article  PubMed  CAS  Google Scholar 

  36. Merten, O.-W., Petres, S., and Couve, E. (1995) A simple serum-free freezing medium for serum-free cultured cells. Biologicals 23, 185–189.

    Article  PubMed  CAS  Google Scholar 

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  1. Genetic Institute, One Burt Road, 01810, Andover, MA

    Martin S. Sinacore

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  1. Martin S. Sinacore
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  2. Denis Drapeau
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  3. S. R. Adamson
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Sinacore, M.S., Drapeau, D. & Adamson, S.R. Adaptation of mammalian cells to growth in serum-free media. Mol Biotechnol 15, 249–257 (2000). https://doi.org/10.1385/MB:15:3:249

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