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. 2006 Aug 5;50(1-3):49–56. doi: 10.1007/s10616-006-9002-y

The role of recombinant proteins in the development of serum-free media

Joanne Keenan 1,, Dermot Pearson 2, Martin Clynes 1
PMCID: PMC3476004  PMID: 19003070

Abstract

Early developments in serum-free media led to a variety of formulations in which components normally provided in serum and required for growth (insulin, transferrin, lipid supplements, trace elements) and poorly defined components (extracts, hydrolysates) were added to defined basal media. These additives were mostly animal-derived. Given recent concerns about TSEs (transmissible spongiform encephalopathies) and other adventitious agents, the drive in media formulations must be towards elimination of animal-origin materials while maintaining cell line productivity. The progress made towards removing animal-derived components and the use of recombinant proteins in serum-free media for mammalian cells is reviewed.

References

  1. Balls P, Park KJ, Barnett B. Production of monoclonal antibodies in serum-free media prepared with bovine transferrin. Art Sci. 1997;16(3):1–4. [Google Scholar]
  2. Barnes D, Sato G. Serum-free cell culture: a unifying approach. Cell. 1980;22:649–655. doi: 10.1016/0092-8674(80)90540-1. [DOI] [PubMed] [Google Scholar]
  3. Barnes DW. Serum-free animal cell culture. Biotechniques. 1987;5:534–541. doi: 10.1038/nbt0687-534. [DOI] [Google Scholar]
  4. Blake D, Svalander P, Jin S, Silversand C, Hamberger L. Protein supplementation of human IVF culture media. J Assist Reprod Gen. 2002;19(3):137–143. doi: 10.1023/A:1014788821965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bradshaw GL, Sato GH, McClure DB, Dubes GR. The growth requirements of BHK-21 in serum-free culture. J Cell Physiol. 1994;114(2):215–221. doi: 10.1002/jcp.1041140211. [DOI] [PubMed] [Google Scholar]
  6. Brands R, Visser J, Medema J, Palache AM, Scharrenburg GJ. Influvac: a safe Madin Darby Canine Kidney (MDCK) cell culture-based influenza vaccine. Dev Biol Stand. 1999;98:93–100. [PubMed] [Google Scholar]
  7. Castle P, Robertson JS. Animal sera, animal sera derivatives and substitutes used in the manufacture of pharmaceuticals. Biologicals. 1998;26:365–368. doi: 10.1006/biol.1998.0165. [DOI] [PubMed] [Google Scholar]
  8. Castro PM, Ison AP, Hayter PM, Bull AT. The macroheterogeneity of recombinant human interferon-gamma produced by Chinese-hamster ovary cells is affected by the protein and lipid content of the culture medium. Biotechnol Appl Biochem. 1995;21(Pt 1):87–100. [PubMed] [Google Scholar]
  9. Chu L, Robinson DK. Industrial choices for protein production by large-scale cell culture. Curr Opin Biotechnol. 2001;12:180–187. doi: 10.1016/S0958-1669(00)00197-X. [DOI] [PubMed] [Google Scholar]
  10. Cinatl J, Jr, Cinatl J, Rabenau H, Rapp J, Kornhumer B, Doeer HW. Protein-free culture of Vero cells: a substrate for replication of human pathogenic viruses. Cell Biol Int Rep. 1993;17:885–895. doi: 10.1006/cbir.1993.1152. [DOI] [PubMed] [Google Scholar]
  11. Congote LF. Extraction of an erythrotropin-like factor from bovine serum albumin (Cohn fraction V) In Vitro Cell Dev Biol. 1987;23(5):361–6. doi: 10.1007/BF02620993. [DOI] [PubMed] [Google Scholar]
  12. Conover CA, Hintz RL, Rosenfeld RG. Comparative effects of somatomedin C and insulin on the metabolism and growth of cultured human fibroblasts. J Cell Physiol. 1985;122:133–141. doi: 10.1002/jcp.1041220120. [DOI] [PubMed] [Google Scholar]
  13. Conrad ME, Umbreit JN, Moore EG, Uzel C, Berry M. Alternate iron transport pathway: mobilferrin and integrin in K562 cells. J Biol Chem. 1994;269(10):7169–7173. [PubMed] [Google Scholar]
  14. Darfler FJ. Preparation and use of lipid microemulsions as nutritional supplements for culturing mammalian cells. In Vitro Cell Dev Biol. 1990;26:779–783. doi: 10.1007/BF02623619. [DOI] [PubMed] [Google Scholar]
  15. Darfler FJ. A protein-free medium for the growth of hybridomas and other cells of the immune system. In Vitro Cell Dev Biol. 1990a;26:769–778. doi: 10.1007/BF02623618. [DOI] [PubMed] [Google Scholar]
  16. Draper JS, Moore HD, Ruban LN, Gokhale PJ, Andrews PW. Culture and characterization of human embryonic stem cells. Stem Cells Dev. 2004;13(4):325–36. doi: 10.1089/scd.2004.13.325. [DOI] [PubMed] [Google Scholar]
  17. Eagle H. Nutrition needs of mammalian cells in tissue culture. Science. 1955;122:501–504. doi: 10.1126/science.122.3168.501. [DOI] [PubMed] [Google Scholar]
  18. Fitzsimmons JS, Sanyal A, Gonzalez C, Fukumoto T, Clemens VR, O’Driscoll SW, Reinholz GG. Serum-free media for periosteal chondrogenesis in vitro. Journal of Orthopaedic Research. 2004;22:716–725. doi: 10.1016/j.orthres.2003.10.020. [DOI] [PubMed] [Google Scholar]
  19. Frazatti-Gallina NM, Mourão-Fuches RM, Paoli RL, Silva MLN, Miyaki C, Valentini EJG, Raw I, Higashi HG. Vero-cell rabies vaccine produced using serum-free medium. Vaccine. 2004;23:511–517. doi: 10.1016/j.vaccine.2004.06.014. [DOI] [PubMed] [Google Scholar]
  20. Gorfien S, Paul B, Walowitz J, Keem R, Biddle W, Jayme D. Growth of NS0 Cells in Protein-Free, Chemically Defined Medium. Biotechnol Prog. 2000;16:682–687. doi: 10.1021/bp000109a. [DOI] [PubMed] [Google Scholar]
  21. Gu X, Xie L, Harmon BJ, Wang DIC. Influence of Primatone RL Supplementation on Sialylation of Recombinant Human Interferon-gamma Produced by Chinese Hamster Ovary cell Culture Using Serum-Free Media. Biotech Bioeng. 1997;56(4):353–341. doi: 10.1002/(SICI)1097-0290(19971120)56:4<353::AID-BIT1>3.0.CO;2-N. [DOI] [PubMed] [Google Scholar]
  22. Gutteridge JMC, Paterson SK, Segal AW, Halliwell B. Inhibition of lipid peroxidation by the iron-binding protein lactoferrin. Biochem J. 1981;199:259–261. doi: 10.1042/bj1990259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ham RG. Formulation of basal nutrition media. In: Barnes DW, Sirbasku DA, Sato GH, editors. Methods for Preparation of media, supplements, and substrata for serum-free animal cell culture, cell culture methods for molecular and cell biology. New York: Liss; 1984. [Google Scholar]
  24. Harvey MB, Kaye PL. Medication of the actions of insulin and insulin-like growth factor-I on pre-implanation mouse embryos in vitro. Mol Reprod Dev. 1992;333:270–275. doi: 10.1002/mrd.1080330306. [DOI] [PubMed] [Google Scholar]
  25. Hayavi S, Halbert GW. Synthetic low-density lipoprotein, a novel biomimetic lipid supplement for serum-free tissue culture. Biotechnol Prog. 2005;21:1262–1268. doi: 10.1021/bp050043p. [DOI] [PubMed] [Google Scholar]
  26. Heidemann R, Zhang C, Qi H, Rule JL, Rozales C, Park S, Chuppa S, Ray M, Michaels J, Konstantinov K, Naveh D. The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells. Cytotechnology. 2000;32:157–167. doi: 10.1023/A:1008196521213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hesse F, Ebel M, Konisch N, Sterlinski R, Kessler W, Wagner R. Comparison of a Production Process in a Membrane-Aerated Stirred Tank and up to 1000-L Airlift Bioreactors Using BHK-21 Cells and Chemically Defined Protein-Free Medium. Biotechnol Prog. 2003;19:833–843. doi: 10.1021/bp0257630. [DOI] [PubMed] [Google Scholar]
  28. Hewlett G, Duvinski MS, Montalto JG. PENTEX EX-CYTE growth enhancement media supplement as a lipoprotein additive for mammalian cell culture. Miles Science J. (Elkhart, IN) 1989;11(1):9–14. [Google Scholar]
  29. Hoffmann C, Goldfines ID, Whittaker J. The metabolic and mitogenic effects of both insulin and insulin-like growth factor are enhanced by transfection of insulin receptors into NIH3T3 fibroblasts. J Biol Chem. 1989;264(15):8606–8611. [PubMed] [Google Scholar]
  30. Hohenblum H, Naschberger S, Katinger H, Mattanovich D (2000) Production of recombinant human trypsinogen in E. Coli and Pichia pastoris_ a comparison of expression systems. Presented at the EFB meeting on recombinant protein production with prokaryotic and eukarotic cells. A comparative view on host physiology. Semmering/A 5-8.10
  31. Jäger V, Lehmann J, Friedl P. Serum-free growth medium for the cultivation of a wide spectrum of mammalian cells in stirred bioreactors. Cytotechnology. 1988;1:319–329. doi: 10.1007/BF00365077. [DOI] [PubMed] [Google Scholar]
  32. Jayme DW. An animal origin perspective of common constituents of serum-free medium formulations. Dev Biol Stand. 1999;99:181–7. [PubMed] [Google Scholar]
  33. Johnson EW, Meunier SF, Roy CJ, Parenteau NL. Serial cultivation of normal human keratinocytes: a defined system for studying the regulation of growth and differentiation. In Vitro Cell Dev Biol. 1992;28a:429–435. doi: 10.1007/BF02634047. [DOI] [PubMed] [Google Scholar]
  34. Jonas HA, Harrison LC. The human placenta contains two distinct binding and immunoreactive species of insulin-like growth factor-I receptors. J Biol Chem. 1985;260(4):2288–2294. [PubMed] [Google Scholar]
  35. Kane M (1990) Control of growth in pre-implantation embryos. I.J.M.S January, 17–21
  36. Keenan J, Clynes M. Replacement of transferrin by simple iron compounds for MDCK cells grown and subcultured in serum-free medium. In Vitro Cell Dev Biol. 1996;32(8):451–453. doi: 10.1007/BF02723044. [DOI] [PubMed] [Google Scholar]
  37. Kim B-S, Yoo SP, Park H-W. Tissue engineering of cartilage with chondrocytes cultured in a chemically-defined, serum-free medium. Biotechnol Lett. 2004;26:709–712. doi: 10.1023/B:BILE.0000024093.94151.d8. [DOI] [PubMed] [Google Scholar]
  38. Kim DY, Lee CL, Chang HN, Oh DJ. Effects of supplementation of various medium components on Chinese hamster ovary cell cultures producing recombinant antibody. Cytotechnology. 2005;47:37–49. doi: 10.1007/s10616-005-3775-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Kovar J. Insoluble iron compound is able to stimulate growth of cultured cells. In Vitro Cell Dev Biol. 1990;26(11):1026–1027. doi: 10.1007/BF02624434. [DOI] [PubMed] [Google Scholar]
  40. Kovar J., Franek F. Iron compounds at high concentrations enable hybridoma growth in a protein-free medium. Biotechnol Lett. 1987;9(4):259–264. doi: 10.1007/BF01027160. [DOI] [Google Scholar]
  41. Kretzmer G. Industrial processes with animal cells. Appl Microbiol Biotechnol. 2002;59:135–142. doi: 10.1007/s00253-002-0991-y. [DOI] [PubMed] [Google Scholar]
  42. Lai DZ, Weng SJ, Qi LQ, Yu CM, Fu L, Yu T, Chen W. Construction of two robust CHO cell lines resistant to apoptosis and adapted to protein-free medium by over-expression of Igf-1/bcl-2 or bcl-2/cyclin E genes. Sheng Wu Gong Cheng Xue Bao. 2004;20(1):66–72. [PubMed] [Google Scholar]
  43. Lammers R, Gray A, Schlessinger J, Ullrich A. Differential signalling potential of insulin and IGF-I receptor cytoplasmic domains. EMBO J. 1989;8(5):1369–1375. doi: 10.1002/j.1460-2075.1989.tb03517.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Laskey J, Webb I, Schulman H, Ponka P. Evidence that transferrin supports cell proliferation by supplying iron for DNA synthesis. Exp Cell Res. 1988;176:87–95. doi: 10.1016/0014-4827(88)90123-1. [DOI] [PubMed] [Google Scholar]
  45. Litwin J. The growth of Vero cells in suspension as cell-aggregates in serum-free media. Cytotechnology. 1992;10:169–174. doi: 10.1007/BF00570893. [DOI] [PubMed] [Google Scholar]
  46. Liu CH, Chu IM, Hwang SM. Factorial designs combined with steepest ascent method to optimise serum-free media for CHO cells. Enz Microb Technol. 2001;28:314–321. doi: 10.1016/S0141-0229(00)00346-X. [DOI] [PubMed] [Google Scholar]
  47. Loo D, Rawson C, Schmitt M., Lindburg K., Barnes D. Glucocorticoid and thyroid hormones inhibit proliferation of SF mouse embryo cells. J Cell Physiol. 1990;142:210–217. doi: 10.1002/jcp.1041420126. [DOI] [PubMed] [Google Scholar]
  48. Merten O-W, Wu R, Crainic R. Evaluation of the serum-free medium MDSS2 for the production of polio virus on Vero cells in bioreactors. Cytotechnology. 1996;25:35–44. doi: 10.1023/A:1007999313566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Merten O-W, Kallel H, Manuguerra J-C, Tardy-Panit M, Crainic R, Delpeyroux F, Vander Werf S, Perrin P. The new medium MDSS2N, free of any animal protein supports cell growth and production of various viruses. Cytotechnology. 1999;30:191–201. doi: 10.1023/A:1008021317639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Merten O-W. Safety issues of animal products used in serum-free media. Dev Biol Stand. 1999;99:167–80. [PubMed] [Google Scholar]
  51. Merten O-W. Cell detachment. In: Spier RE, editor. Encyclopedia of cell technology. New York USA: J Wiley and Sons Inc; 1999a. pp. 351–365. [Google Scholar]
  52. Merten O-W. Development of serum-free media for cell growth and production of viruses/viral vaccines—safety issues of animal products used in serum-free media. Dev Biol. 2002;111:235–259. [PubMed] [Google Scholar]
  53. Merten O-W. Virus contamination of cell cultures—A biotechnological view. Cytotechnology. 2002a;39:91–116. doi: 10.1023/A:1022969101804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Möbest D, Mertelsmann R, Henschler R. Serum-free ex vivo expansion of CD34(+) hematopoietic progenitor cells. Biotechnol Bioeng. 1998;60(3):341–7. doi: 10.1002/(SICI)1097-0290(19981106)60:3<341::AID-BIT10>3.0.CO;2-P. [DOI] [PubMed] [Google Scholar]
  55. Mols J, Peeters-Joris C, Wattiez R, Agathos SN, Schneider YJ. Recombinant interferon-T secreted by Chinese hamster ovary-320 cells cultivated in suspension in protein-free media is protected against extracellular proteolysis by the expression of natural protease inhibitors and by the addition of plant protein hydrolysates to the culture medium. In Vitro Cell Dev Biol. 2005;41(3–4):83–91. doi: 10.1290/0411075.1. [DOI] [PubMed] [Google Scholar]
  56. Morris AE, Schmid J. Effects of insulin and long R3 on serum-free Chinese hamster ovary cell cultures expressing two recombinant proteins. Biotechnol Progr. 2000;16:693–697. doi: 10.1021/bp0000914. [DOI] [PubMed] [Google Scholar]
  57. Moss AM, Livingston JN. Distinct ß-subunits are present in hybrid insulin-like growth factor-I receptors in the central nervous system. Biochem Journal. 1993;294(3):685–692. doi: 10.1042/bj2940685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Okamoto T, Tani R, Yabumoto M, Sakamoto A, Takada K, Sato GH, Sato JD. Effects of insulin and transferrin on the generation oflymphokine-activated killer cells in serum-free medium. J Immunol Methods. 1996;195:7–14. doi: 10.1016/0022-1759(96)00081-6. [DOI] [PubMed] [Google Scholar]
  59. Palache AM, Brands R, Scharrenburg GJM. Immunogenicity and reactogenicity of influenza subunit vaccines produced in MDCK cells or fertilized chicken eggs. J Infect Dis. 1997;176:S20–S23. doi: 10.1086/514169. [DOI] [PubMed] [Google Scholar]
  60. Perrin P, Malhusudana S, Gontier-Jallet C, Petres S, Tordo N, Merten OW. An experimental rabies vaccine produced with a new BHK-21 suspension culture process: use of serum-free medium and perfusion-reactor system. Vaccine. 1995;13:1244–1250. doi: 10.1016/0264-410X(94)00022-F. [DOI] [PubMed] [Google Scholar]
  61. Richardson DR, Baker E. Two saturable mechanisms of iron uptake from transferrin in human melanoma cells: the effect of transferrin concentration, chelators, and metabolic probes on transferrin and iron uptake. J Cell Physiol. 1994;161(1):160–8. doi: 10.1002/jcp.1041610119. [DOI] [PubMed] [Google Scholar]
  62. Saad B, Schawalder H, Maier P. Crude liver membrane fractions as substrate preserve liver-specific functions in long-term, serum-free rat hepatocyte cultures. In Vitro Cell Dev Biol. 1993;29a(1):32–40. doi: 10.1007/BF02634369. [DOI] [PubMed] [Google Scholar]
  63. Sanders EJ, Cheung E. Transferrin and iron requirements of embryonic mesoderm cells cultured in hydrated collagen matrices. In Vitro Cell Dev Biol. 1988;24(6):581–587. doi: 10.1007/BF02629094. [DOI] [PubMed] [Google Scholar]
  64. Savonniere S, Zeghari N, Miccoli L, Muller S, Maugras M, Donner M. Effects of lipid supplementation of culture media on cell growth, antibody production, membrane structure and dynamics in two hybridomas. J Biotechnol. 1996;18(48):161–173. doi: 10.1016/0168-1656(96)01392-2. [DOI] [PubMed] [Google Scholar]
  65. Schröder M, Matischak K, Friedl P. Serum- and protein-free media formulations for the Chinese hamster ovary cell line DUKXB11. J Biotechnol. 2004;108:279–292. doi: 10.1016/j.jbiotec.2003.12.005. [DOI] [PubMed] [Google Scholar]
  66. Shinmoto H, Dosako S, Taneya S. Long-term culture of mouse hybridoma HB8852 cells in a protein-free medium. Biotechnol Lett. 1988;10(10):683–689. doi: 10.1007/BF01025282. [DOI] [Google Scholar]
  67. Spens E, Häggström L. Defined protein-free NS0 myeloma cell cultures: stimulation of proliferation by conditioned medium factors. Biotechnol Prog. 2005;21(1):87–95. doi: 10.1021/bp049822g. [DOI] [PubMed] [Google Scholar]
  68. Sun YH, Lim SW, Chung JY, Lee GM. Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells. Appl Microbiol Biotechnol. 2004;63:527–536. doi: 10.1007/s00253-003-1389-1. [DOI] [PubMed] [Google Scholar]
  69. Tartare S, Mothe I, Kowalskichauvel A, Breittmayer JP, Ballotti R, Obberghen E. Signal transduction by a chimeric insulin-like growth factor-I (IGF-I) receptor having the carboxyl-terminal domain of the insulin receptor. J Biol Chem. 1994;269(15):11449–11455. [PubMed] [Google Scholar]
  70. Tang X-h, Shay NF. Zinc Has an Insulin-Like Effect on Glucose Transport Mediated by Phosphoinositol-3-Kinase and Akt in 3T3-L1 Fibroblasts and Adipocytes. J Nutri. 2001;131:1414–1420. doi: 10.1093/jn/131.5.1414. [DOI] [PubMed] [Google Scholar]
  71. Tigyi G, Miledi R. Lysophosphatidates bound to serum albumin activate membrane currents in XenopusOocytes and Neurite Retraction in PC12 Pheochromocytoma cells. J Biol Chem. 1992;267:21360–21367. [PubMed] [Google Scholar]
  72. Tsao M, sanders GHS, Grisham JW. Regulation of cultured hepatic epithelial cells by transferrin. Exp Cell Res. 1987;171:52–62. doi: 10.1016/0014-4827(87)90250-3. [DOI] [PubMed] [Google Scholar]
  73. Vyhlidal C, Li X, Safe S. Estrogen regulation of transferrin gene expression in MCF-7 human breast cancer cells. J Mol Endocrinol. 2002;29(3):305–17. doi: 10.1677/jme.0.0290305. [DOI] [PubMed] [Google Scholar]
  74. Werner RG. Economic aspects of commercial manufacture of biopharmaceuticals. J Biotechnol. 2004;113:171–182. doi: 10.1016/j.jbiotec.2004.04.036. [DOI] [PubMed] [Google Scholar]
  75. Wong VVT, Kah WH, Yap MGS. Evaluation of insulin-mimetic trace elements as insulin replacements in mammalian cell culture. Cytotechnology. 2004;45:107–115. doi: 10.1007/s10616-004-6173-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Yao CL, Liu CH, Chu IM, Hsieh TB, Hwang SM. Factorial designs combined with the steepest ascent method to optimize serum-free media for ex vivo expansion of human hematopoietic progenitor cells. Enzyme Microb Technol. 2003;33:343–352. doi: 10.1016/S0141-0229(03)00144-3. [DOI] [Google Scholar]
  77. Yelian FD, Edgeworth NA, Li-Jin Dong L-J, Chung AE, Armant DR. Recombinant entactin promotes mouse primary trophoblast cell adhesion and migration through the Arg-Gly-Asp (RGD) recognition sequence. J Cell Biol. 1993;121:923–929. doi: 10.1083/jcb.121.4.923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Zhang J, Robinson D. Development of animal-free, protein-free and chemically-defined media for NSO cell culture. Cytotechnology. 2005;48:59–74. doi: 10.1007/s10616-005-3563-z. [DOI] [PMC free article] [PubMed] [Google Scholar]

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