, 50:49 | Cite as

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

  • Joanne KeenanEmail author
  • Dermot Pearson
  • Martin Clynes


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.


Transferrin Bovine Spongiform Encephalopathy Madin Darby Canine Kidney Madin Darby Canine Kidney Cell Adventitious Agent 
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.


  1. Balls P, Park KJ, Barnett B (1997) Production of monoclonal antibodies in serum-free media prepared with bovine transferrin. Art Sci 16(3):1–4Google Scholar
  2. Barnes D, Sato G (1980) Serum-free cell culture: a unifying approach. Cell 22:649–655CrossRefGoogle Scholar
  3. Barnes DW (1987) Serum-free animal cell culture. Biotechniques 5:534–541CrossRefGoogle Scholar
  4. Blake D, Svalander P, Jin S, Silversand C, Hamberger L (2002) Protein supplementation of human IVF culture media. J␣Assist Reprod Gen 19(3):137–143CrossRefGoogle Scholar
  5. Bradshaw GL, Sato GH, McClure DB, Dubes GR (1994) The growth requirements of BHK-21 in serum-free culture. J Cell Physiol 114(2):215–221CrossRefGoogle Scholar
  6. Brands R, Visser J, Medema J, Palache AM, van Scharrenburg GJ (1999) Influvac: a safe Madin Darby Canine Kidney (MDCK) cell culture-based influenza vaccine. Dev Biol Stand 98:93–100Google Scholar
  7. Castle P and Robertson JS (1998) Animal sera, animal sera derivatives and substitutes used in the manufacture of pharmaceuticals. Biologicals 26:365–368CrossRefGoogle Scholar
  8. Castro PM, Ison AP, Hayter PM, Bull AT (1995) 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 21 (Pt 1):87–100Google Scholar
  9. Chu L and Robinson DK (2001) Industrial choices for protein production by large-scale cell culture. Curr Opin Biotechnol 12:180–187CrossRefGoogle Scholar
  10. Cinatl J Jr, Cinatl J, Rabenau H, Rapp J, Kornhumer B, Doeer HW (1993) Protein-free culture of Vero cells: a substrate for replication of human pathogenic viruses. Cell Biol Int Rep 17:885–895CrossRefGoogle Scholar
  11. Congote LF (1987) Extraction of an erythrotropin-like factor from bovine serum albumin (Cohn fraction V). In Vitro Cell Dev Biol 23(5):361–6CrossRefGoogle Scholar
  12. Conover CA, Hintz RL and Rosenfeld RG (1985) Comparative effects of somatomedin C and insulin on the metabolism and growth of cultured human fibroblasts. J Cell Physiol 122:133–141CrossRefGoogle Scholar
  13. Conrad ME, Umbreit JN, Moore EG, Uzel C and Berry M (1994) Alternate iron transport pathway: mobilferrin and␣integrin in K562 cells. J Biol Chem 269(10):7169–7173Google Scholar
  14. Darfler FJ (1990) Preparation and use of lipid microemulsions as nutritional supplements for culturing mammalian cells. In Vitro Cell Dev Biol 26:779–783CrossRefGoogle Scholar
  15. Darfler FJ (1990a) A protein-free medium for the growth of hybridomas and other cells of the immune system. In Vitro Cell Dev Biol 26:769–778CrossRefGoogle Scholar
  16. Draper JS, Moore HD, Ruban LN, Gokhale PJ, Andrews PW (2004) Culture and characterization of human embryonic stem cells. Stem Cells Dev 13(4):325–36CrossRefGoogle Scholar
  17. Eagle H (1955) Nutrition needs of mammalian cells in tissue culture. Science 122:501–504CrossRefGoogle Scholar
  18. Fitzsimmons JS, Sanyal A, Gonzalez C, Fukumoto T, Clemens VR, O’Driscoll SW, Reinholz GG (2004) Serum-free media for periosteal chondrogenesis in vitro. Journal of Orthopaedic Research 22:716–725CrossRefGoogle Scholar
  19. Frazatti-Gallina NM, Mourão-Fuches RM, Paoli RL, Silva MLN, Miyaki C, Valentini EJG, Raw I and Higashi HG (2004) Vero-cell rabies vaccine produced using serum-free medium. Vaccine 23:511–517CrossRefGoogle Scholar
  20. Gorfien S, Paul B, Walowitz J, Keem R, Biddle W, and Jayme D (2000) Growth of NS0 Cells in Protein-Free, Chemically Defined Medium. Biotechnol Prog 16:682–687CrossRefGoogle Scholar
  21. Gu X, Xie L, Harmon BJ, Wang DIC (1997) 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 56(4):353–341CrossRefGoogle Scholar
  22. Gutteridge JMC, Paterson SK, Segal AW and Halliwell B (1981) Inhibition of lipid peroxidation by the iron-binding protein lactoferrin. Biochem J 199:259–261Google Scholar
  23. Ham RG (1984) Formulation of basal nutrition media. In: Barnes DW, Sirbasku DA, Sato GH (eds) Methods for preparation of media, supplements, and substrata for serum-free animal cell culture, cell culture methods for molecular and cell biology, vol. 1. Liss, New YorkGoogle Scholar
  24. Harvey MB, Kaye PL (1992) Medication of the actions of insulin and insulin-like growth factor-I on pre-implantation mouse embryos in vitro. Mol Reprod Dev 333:270–275CrossRefGoogle Scholar
  25. Hayavi S, Halbert GW (2005) Synthetic low-density lipoprotein, a novel biomimetic lipid supplement for serum-free tissue culture. Biotechnol Prog 21:1262–1268CrossRefGoogle Scholar
  26. Heidemann R, Zhang C, Qi H, Rule JL, Rozales C, Park S, Chuppa S, Ray M, Michaels J, Konstantinov K, Naveh D (2000) The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells. Cytotechnology 32:157–167CrossRefGoogle Scholar
  27. Hesse F, Ebel M, Konisch N, Sterlinski R, Kessler W and Wagner R (2003). 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 19:833–843CrossRefGoogle Scholar
  28. Hewlett, G, Duvinski MS and Montalto JG (1989) PENTEX EX-CYTE growth enhancement media supplement as a lipoprotein additive for mammalian cell culture. Miles Science J. (Elkhart, IN) 11(1):9–14Google Scholar
  29. Hoffmann C, Goldfines ID and Whittaker J (1989) 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 264(15):8606–8611Google 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 5-8.10Google Scholar
  31. Jäger V, Lehmann J, Friedl P (1988) Serum-free growth medium for the cultivation of a wide spectrum of mammalian cells in stirred bioreactors. Cytotechnology 1:319–329CrossRefGoogle Scholar
  32. Jayme DW (1999) An animal origin perspective of common constituents of serum-free medium formulations. Dev Biol Stand 99:181–7Google Scholar
  33. Johnson EW, Meunier SF, Roy CJ and Parenteau NL (1992) Serial cultivation of normal human keratinocytes: a defined system for studying the regulation of growth and differentiation. In Vitro Cell Dev Biol 28a:429–435CrossRefGoogle Scholar
  34. Jonas HA and Harrison LC (1985) The human placenta contains two distinct binding and immunoreactive species of insulin-like growth factor-I receptors. J Biol Chem 260(4):2288–2294Google Scholar
  35. Kane M (1990) Control of growth in pre-implantation embryos. I.J.M.S January, 17–21Google Scholar
  36. Keenan J, Clynes M (1996) Replacement of transferrin by simple iron compounds for MDCK cells grown and subcultured in serum-free medium. In Vitro Cell Dev Biol 32(8):451–453CrossRefGoogle Scholar
  37. Kim B-S, Yoo SP, Park H-W (2004) Tissue engineering of cartilage with chondrocytes cultured in a chemically-defined, serum-free medium. Biotechnol Lett 26:709–712CrossRefGoogle Scholar
  38. Kim DY, Lee CL, Chang HN, Oh DJ (2005) Effects of supplementation of various medium components on Chinese hamster ovary cell cultures producing recombinant antibody. Cytotechnology 47:37–49CrossRefGoogle Scholar
  39. Kovar J (1990) Insoluble iron compound is able to stimulate growth of cultured cells. In Vitro Cell Dev Biol 26(11):1026–1027CrossRefGoogle Scholar
  40. Kovar J. and Franek F (1987) Iron compounds at high concentrations enable hybridoma growth in a protein-free medium. Biotechnol Lett 9(4):259–264CrossRefGoogle Scholar
  41. Kretzmer G (2002) Industrial processes with animal cells. Appl Microbiol Biotechnol 59:135–142CrossRefGoogle Scholar
  42. Lai DZ, Weng SJ, Qi LQ, Yu CM, Fu L, Yu T, Chen W (2004) 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 20(1):66–72Google Scholar
  43. Lammers R, Gray A, Schlessinger J and Ullrich A (1989) Differential signalling potential of insulin and IGF-I receptor cytoplasmic domains. EMBO J 8(5):1369–1375Google Scholar
  44. Laskey J, Webb I, Schulman H and Ponka P (1988) Evidence that transferrin supports cell proliferation by supplying iron for DNA synthesis. Exp Cell Res 176:87–95CrossRefGoogle Scholar
  45. Litwin J (1992) The growth of Vero cells in suspension as cell-aggregates in serum-free media. Cytotechnology 10:169–174CrossRefGoogle Scholar
  46. Liu CH, Chu IM, Hwang SM (2001) Factorial designs combined with steepest ascent method to optimise serum-free media for CHO cells. Enz Microb Technol 28:314–321CrossRefGoogle Scholar
  47. Loo D, Rawson C, Schmitt M, Lindburg K, Barnes D (1990) Glucocorticoid and thyroid hormones inhibit proliferation of SF mouse embryo cells. J Cell Physiol 142:210–217CrossRefGoogle Scholar
  48. Merten O-W, Wu R, Crainic R (1996) Evaluation of the serum-free medium MDSS2 for the production of polio virus on Vero cells in bioreactors. Cytotechnology 25:35–44CrossRefGoogle Scholar
  49. Merten O-W, Kallel H, Manuguerra J-C, Tardy-Panit M, Crainic R, Delpeyroux F, van der Werf S, Perrin P (1999) The new medium MDSS2N, free of any animal protein supports cell growth and production of various viruses. Cytotechnology 30:191–201CrossRefGoogle Scholar
  50. Merten O-W (1999) Safety issues of animal products used in serum-free media. Dev Biol Stand 99:167–80Google Scholar
  51. Merten O-W (1999a) Cell detachment. In: Spier RE (ed) Encyclopedia of cell technology. J Wiley and Sons Inc, New York USA, pp 351–365Google Scholar
  52. Merten O-W (2002) 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 111:235–259Google Scholar
  53. Merten O-W (2002a) Virus contamination of cell cultures—A biotechnological view. Cytotechnology 39:91–116CrossRefGoogle Scholar
  54. Möbest D, Mertelsmann R, Henschler R (1998) Serum-free ex vivo expansion of CD34(+) hematopoietic progenitor cells. Biotechnol Bioeng 60(3):341–7CrossRefGoogle Scholar
  55. Mols J, Peeters-Joris C, Wattiez R, Agathos SN, Schneider YJ (2005) 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 41(3–4):83–91CrossRefGoogle Scholar
  56. Morris AE and Schmid J (2000) Effects of insulin and long R3 on serum-free Chinese hamster ovary cell cultures expressing two recombinant proteins. Biotechnol Progr 16:693–697CrossRefGoogle Scholar
  57. Moss AM and Livingston JN (1993) Distinct ß-subunits are present in hybrid insulin-like growth factor-I receptors in the central nervous system. Biochem Journal 294(3):685–692Google Scholar
  58. Okamoto T, Tani R, Yabumoto M, Sakamoto A, Takada K, Sato GH, Sato JD (1996) Effects of insulin and transferrin on the generation oflymphokine-activated killer cells in serum-free medium. J Immunol Methods 195:7–14CrossRefGoogle Scholar
  59. Palache AM, Brands R, van Scharrenburg GJM (1997) Immunogenicity and reactogenicity of influenza subunit vaccines produced in MDCK cells or fertilized chicken eggs. J Infect Dis 176:S20–S23CrossRefGoogle Scholar
  60. Perrin P, Malhusudana S, Gontier-Jallet C, Petres S, Tordo N, Merten OW (1995) An experimental rabies vaccine produced with a new BHK-21 suspension culture process: use of serum-free medium and perfusion-reactor system. Vaccine 13:1244–1250CrossRefGoogle Scholar
  61. Richardson DR, Baker E (1994) 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 161(1):160–8CrossRefGoogle Scholar
  62. Saad B, Schawalder H and Maier P (1993) Crude liver membrane fractions as substrate preserve liver-specific functions in long-term, serum-free rat hepatocyte cultures. In Vitro Cell Dev Biol 29a(1):32–40CrossRefGoogle Scholar
  63. Sanders EJ, Cheung E (1988) Transferrin and iron requirements of embryonic mesoderm cells cultured in hydrated collagen matrices. In Vitro Cell Dev Biol 24(6):581–587CrossRefGoogle Scholar
  64. Savonniere S, Zeghari N, Miccoli L, Muller S, Maugras M, Donner M (1996) Effects of lipid supplementation of culture media on cell growth, antibody production, membrane structure and dynamics in two hybridomas. J Biotechnol 18(48):161–173CrossRefGoogle Scholar
  65. Schröder M, Matischak K, Friedl P (2004) Serum- and protein-free media formulations for the Chinese hamster ovary cell line DUKXB11. J Biotechnol 108:279–292CrossRefGoogle Scholar
  66. Shinmoto H, Dosako S, Taneya S (1988) Long-term culture of mouse hybridoma HB8852 cells in a protein-free medium. Biotechnol Lett 10(10):683–689CrossRefGoogle Scholar
  67. Spens E and Häggström L (2005) Defined protein-free NS0 myeloma cell cultures: stimulation of proliferation by conditioned medium factors. Biotechnol Prog 21(1):87–95CrossRefGoogle Scholar
  68. Sun YH, Lim SW, Chung JY, Lee GM (2004) 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 63:527–536CrossRefGoogle Scholar
  69. Tartare S, Mothe I, Kowalskichauvel A, Breittmayer JP, Ballotti R, Van Obberghen E (1994) 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 269(15):11449–11455Google Scholar
  70. Tang X-h and Shay NF (2001) Zinc Has an Insulin-Like Effect on Glucose Transport Mediated by Phosphoinositol-3-Kinase and Akt in 3T3-L1 Fibroblasts and Adipocytes. J␣Nutri 131:1414–1420Google Scholar
  71. Tigyi G, Miledi R (1992) Lysophosphatidates bound to serum albumin activate membrane currents in XenopusOocytes and Neurite Retraction in PC12 Pheochromocytoma cells. J Biol Chem 267:21360–21367Google Scholar
  72. Tsao M, Sanders GHS, Grisham JW (1987) Regulation of cultured hepatic epithelial cells by transferrin. Exp Cell Res 171:52–62CrossRefGoogle Scholar
  73. Vyhlidal C, Li X, Safe S (2002) Estrogen regulation of transferrin gene expression in MCF-7 human breast cancer cells. J Mol Endocrinol 29(3):305–17CrossRefGoogle Scholar
  74. Werner RG (2004) Economic aspects of commercial manufacture of biopharmaceuticals. J Biotechnol 113:171–182CrossRefGoogle Scholar
  75. Wong VVT, Kah WH, Yap MGS (2004) Evaluation of insulin-mimetic trace elements as insulin replacements in mammalian cell culture. Cytotechnology 45:107–115CrossRefGoogle Scholar
  76. Yao CL, Liu CH, Chu IM, Hsieh TB, Hwang SM (2003) 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 33:343–352CrossRefGoogle Scholar
  77. Yelian FD, Edgeworth NA, Li-Jin Dong L-J, Chung AE, Armant DR (1993) Recombinant entactin promotes mouse primary trophoblast cell adhesion and migration through the Arg-Gly-Asp (RGD) recognition sequence. J Cell Biol 121:923–929CrossRefGoogle Scholar
  78. Zhang J, Robinson D (2005) Development of animal-free, protein-free and chemically-defined media for NSO cell culture. Cytotechnology 48:59–74CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  1. 1.National Institute for Cellular BiotechnologyDublin City UniversityDublinIreland
  2. 2.Delta Biotechnology Ltd.NottinghamUK

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