Abstract
The dihydrofolate reductase-deficient Chinese hamster ovary cell line, DXB11-CHO, commonly used as a host cell for the production of recombinant proteins requires 7.5% serum-supplementation for optimal growth. Regulatory issues surrounding the use of serum in clinical production processes and the direct and indirect costs of using serum in large-scale production and recovery processes have triggered efforts to derive serum-independent host cell lines. We have successfully isolated a serum-free host that we named Veggie-CHO. Veggie-CHO was generated by adapting DXB11-CHO cells to growth in serum-free media in the absence of exogenous growth factors such as Transferrin and Insulin-like growth factor, which we have previously shown to be essential for growth and viability of DXB11-CHO cells. Veggie-CHO cells have been shown to maintain an average doubling time of 22 hr in continuous growth cultures over a period of three months and have retained the dihydrofolate reductase -deficient phenotype of their parental DXB11 -CHO cells. These properties and the stability of its serum-free phenotype have allowed the use of Veggie-CHO as host cells for transfection and amplified expression of recombinant proteins. We describe the derivation a serum-free recombinant cell line with an average doubling time of 20 hr and specific productivity of 2.5 Units recombinant Flt-3L protein per 10e6 cells per day.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Adamson R (1994) Design and operation of a recombinant mammalian cell manufacturing process for rFVIII. Ann Hematol 68Suppl 3:S9–S14.
Barnes D and Sato G (1980) Methods for growth of cultured cells in serum-free medium. Anal Biochem 102: 255–270.
Bloemkolk J-W and Gray MR (1992) Effect of temperature on hybridoma cell cycle and Mab production. Biotech Bioeng 40: 427–431.
Brasel K, McKenna HJ, Morrissey PJ, Charrier K, Morris AE, Lee CC, Williams DE and Lyman SD (1996) Hematological effects of Flt3 ligand in vivo in mice. Blood 88: 2004–2012.
Chen WY, Bailey EC, McCune SL, Dong JY and Townes TM (1997) Reactivation of silenced, virally transduced genes by inhibitors of histone deacetylase. Proc Natl Acad Sci USA 94: 5798–5803.
Dive C, Gregory CD, Phipps DJ, Evans DL, Milner AE and Wyllie AH (1992) Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry. Biochim Biophys Acta 1133: 275–285.
Evans VJ, Bryant JC, Fioramonti MC, McQuilkin WT, Standford KK and Earle WR (1956) Studies of nutrient media for tissue and cells in vitro. A protein-free chemically defined medium for cultivation of strain L cells. Cancer Res 16: 77–86.
Gandor C, Leist C, Fiechter A and Asselbergs FA (1995) Amplification and expression of recombinant genes in serum-independent Chinese hamster ovary cells. FEBS Lett 377: 290–294.
Hamilton WG and Ham RG (1977) Clonal growth of Chinese hamster cell lines in protein-free media. In Vitro 13: 537–547.
Kaufman RJ (1990) Selection and coamplification of heterologous genes in mammalian cells. Meth Enzymol 185: 537–566.
Kaufman RJ, Davies MV, Wasley LC and Michnick D (1991) Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus. Nucleic Acids Res 19: 4485–4490.
Kaufman RJ, Wasley LC and Dorner AJ (1988) Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. J Biol Chem 263: 6352–6362.
Kruh J (1982) Effects of sodium butyrate, a new pharmacological agent, on cells in culture. Mol Cell Biochem 42: 65–82.
McKenna HJ, de Vries P, Brasel K, Lyman SD and Williams DE (1995) Effect of flt3 ligand on the ex vivo expansion of human CD34+ hematopoietic progenitor cells. Blood 86: 3413–3420.
McKinnon P, Ross M, Wells JR, Ballard FJ and Francis GL (1991) Expression, purification and characterization of secreted recombinant human insulin-like growth factor-I (IGF-I) and the potent variant des(1–3) IGF-I in Chinese hamster ovary cells. J Mol Endocrinol 6: 231–239.
Morris AE, Lee CC, Hodges K, Aldrich TL, Krantz C, Smidt PS and Thomas JN (1997) Expression augmenting sequence element (EASE) isolated from Chinese hamster ovary cells. In: Carrondo MJT, Griffiths B and Moreira JLP (eds.) Animal Cell Technology, Kluwer Academic Publishers, The Netherlands, pp. 529–534.
Sambrook J, Fritsch E and Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold Spring Harbor Press, Cold Spring Harbor.
Sinacore MS, Charlebois TS, 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 SR (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. Biotech Bioeng 52: 518–528.
Sun XM, Snowden RT, Skilleter DN, Dinsdale D, Ormerod MG and Cohen GM (1992) A flow-cytometric method for the separation and quantitation of normal and apoptotic thymocytes. Anal Biochem 204: 351–356.
Thomas JN and Fung V (1994) Comparison of long R3 IGF-1 with insulin in the support of cell growth and recombinant protein expression in CHO cells. In: Spier RE, Griffiths JB and Berthold W (eds.) Animal Cell Technology: Products of Today, Prospects for Tomorrow. ESACT Butterworth-Heinemann).
Urlaub G and Chasin LA (1980) Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci USA 77: 4216–4220.
Weidemann R, Ludwig A and Kretzmer G (1994) Low temperature cultivation — A step towards process optimisation. Cytotechnology 15: 111–116.
Wood CR, Dorner AJ, Morris GE, Alderman EM, Wilson D, O’Hara Jr., RM and Kaufman RJ (1990) High level synthesis of immunoglobulins in Chinese hamster ovary cells. J Immunol 145: 3011–3016.
Wyatt DE and Doak J (1995) Performance characteristics of a protein-free medium for mammalian suspension cells. In: Beuvery EC, Griffiths JB and Zeijlemaker WP (eds.) Animal Cell Technology: Developments Towards the 21st Century, Kluwer Academic Publishers, The Netherlands, pp. 349–356.
Yao A, Huang EW and Rubin H (1991) Growth in high serum concentration leads to rapid deadaptation of cells previously adapted to growth in an extremely low concentration of serum. Proc Natl Acad Sci USA 88: 9422–9425.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1998 Springer Science+Business Media Dordrecht
About this chapter
Cite this chapter
Rasmussen, B., Davis, R., Thomas, J., Reddy, P. (1998). Isolation, characterization and recombinant protein expression in Veggie-CHO: A serum-free CHO host cell line. In: Betenbaugh, M.J., Chalmers, J.J., Arathoon, R., Chaplen, F.W.R., Mastrangelo, A.J. (eds) Cell Culture Engineering VI. Current Applications of Cell Culture Engineering, vol 3. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-4786-6_4
Download citation
DOI: https://doi.org/10.1007/978-94-011-4786-6_4
Publisher Name: Springer, Dordrecht
Print ISBN: 978-94-010-6011-0
Online ISBN: 978-94-011-4786-6
eBook Packages: Springer Book Archive