Summary
A protein-free medium, termed ABC, has been developed which essentially eliminates the need for serum proteins. ABC supports the long-term growth of murine hybridomas as well as other transformed cells of the immune system. The requirement of hybridoma growth for transferrin has been met by substituting the soluble organo-iron compound, sodium nitroprusside. Substantial improvement in the growth of hybridomas was afforded by the inclusion of 18 trace elements complexed to disodium ethylene diaminetetraacetate (EDTA). The medium was further improved by the inclusion of components not found in Ham's F12 medium or by raising the concentrations of existing low molecular weight components. Murine hybridomas can be cultured routinely in this protein-free medium in an anchorage-independent manner with doubling times generally under 24 h. Visualized on electrophoretic gels, levels of monoclonal antibody taken from those cultures often exceeded 80% of the total protein. The medium was also able to support the growth of HuT 78 and H9 cells as well as certain other transformed cells of the immune system. In addition, normal human peripheral blood lymphocytes, activated with phytohemagglutinin and cultured with 50 U/ml recombinant interleukin 2, could be grown for 2 wk with a 50-fold expansion over input cell number.
Similar content being viewed by others
References
Barnes, D.; Sato, G. Serum-free cell culture: a unifying approach. Cell 22: 649–660; 1980.
Basset, P.; Zwiller, J.; Ulrich, G. L., et al. Guanylate cyclase activators hemin and sodium nitroprusside stimulate the growth of transformed cells in serum-free medium. Hormonally defined media. New York: Springer-Verlag; 1983: 219–221.
Bettger, W. J.; Boyce, S. T.; Walthall, B. J., et al. Rapid clonal growth and serial passage of human diploid fibroblasts in a lipid-enriched synthetic medium supplemented with epidermal growth factor, insulin and dexamethasone. Proc. Natl. Acad. Sci. USA 78: 5588–5592; 1981.
Breitman, T. R.; Collins, S. J.; Keene, B. R. Replacement of serum by insulin and transferrin supports growth and differentiation of the human promyelocytic cell line, HL-60. J. Immunol. Methods 39: 369–375; 1980.
Breitman, R. R.; Selonick, S. E.; Collins, S. J. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc. Natl. Acad. Sci. USA 77: 2936–2940; 1980.
Brock, J. H.; Stevenson, J. Replacement of transferrin in serum-free cultures of mitogen-stimulated mouse lymphocytes by a lipophilic iron chelaton. Immunol. Lett. 15: 23–25; 1987.
Brown, R. L.; Griffith, R. L.; Ruscetti, F. W., et al. Modulation of interleukin 2 release from a primate lymphoid cell line in serum-free and serum-containing media. Cell. Immunol. 92: 14–21; 1985.
Chang, T. H.; Steplewski, Z.; Koprowski, H. Production of monoclonal antibodies in serum free medium. J. Immunol. Methods 39: 369–375; 1980.
Cleveland, W. L.; Wood, I.; Erlanger, B. F. Routine large-scale production of monoclonal antibodies in a protein-free culture medium. J. Immunol. Methods 56: 221–234; 1983.
Cole, S. P. C.; Vreeken, E. H.; Mirski, S. E. L., et al. Growth of human × human hybridomas in protein-free medium supplemented with ethanolamine. J. Immunol. Methods 97: 29–33; 1987.
Cuthbert, J. A.; Lipsky, P. E. Identification of low density lipoprotein receptor abnormalities by assaying functional receptors on proliferating lymphocytes. Arteriosclerosis 9: 143–149; 1989.
Darfler, F. J.; Insel, P. A. Clonal growth of lymphoid cells in serum-free media requires elimination of H2O2 toxicity. J. Cell. Physiol. 115: 31–36; 1979.
Darfler, F. J.; Insel, P. A. Serum-free culture of resting, PHA-stimulated, and transformed lymphoid cells, including hybridomas. Exp. Cell Res. 138: 287–295; 1982.
Diem, K.; Lentner, C., editors. Scientific tables, 7th ed. Ardsley, NY: Pharmaceuticals; 1970: 17–750.
Gauwerky, C. C.; Golde, D. W. Lithium enhances growth of human leukaemia cells in vitro. Br. J. Haematol 51: 431–438; 1982.
Ham, R. G. Importance of the basal nutrient medium in the design of hormonally defined media. In: Sato, G. H., ed. Growth of cells in hormonally defined media. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1982: 39–55.
Ham, R. G. Clonal growth of mammalian cells in a chemically defined, synthetic medium. Proc. Natl. Acad. Sci. USA 53: 288–293; 1965.
Innes, G. K.; Fuller, B. J.; Hobbs, K. E. F. Lipid peroxidation in hepatocyte cell cultures; modulation by free radical scavengers and iron. In Vitro Cell. Dev. Biol. 24: 126–132; 1988.
Kovar, J.; Franek, F. Iron compounds at high concentrations enable hybridoma growth in a protein-free medium. Biotech. Lett. 9: 259–264; 1987.
Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277: 680–683; 1970.
Leshem, B.; Cohen, I.; Sherman, L., et al. Typing of HLA class II and class I antigens using PHA-activated, IL-2 propagated T lymphocytes. J. Immunol. Methods 111: 131–135; 1988.
Mather, J.; Kaczarowski, F.; Gabler, R., et al. Effects of water purity and addition of common water contaminants on the growth of cells in serum-free media. Biotechniques 4: 56–63; 1986.
Murakami, H.; Masui, H.; Sato, G. H., et al. Growth of hybridoma cells in serum-free medium: ethanolamine is an essential component. Proc. Natl. Acad. Sci. USA 79: 1158–1162; 1982.
Shacter, E. Serum-free medium for growth factor-dependent and independent plasmacytomas and hybridomas. J. Immunol. Methods 99: 259–270; 1987.
Schneider, Y-J. Optimisation of hybridoma cell growth and monoclonal antibody secretion in a chemically defined, serum-and protein-free culture medium. J. Immunol. Methods 116: 65–77; 1989.
Shive, W.; Pinkerton, F.; Humphreys, J., et al. Development of a chemically defined serum- and protein-free medium for growth of human peripheral lymphocytes. Proc. Natl. Acad. Sci. USA 83: 9–13; 1986.
Spierenburg, G. T.; Oerlemans, F. T. J. J.; van Laarhoven, J. P. R. M., et al. Phototoxicity ofN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid-buffered culture media for human leukemic cells lines. Can. Res. 44: 2253–2254; 1984.
Taetle, R.; Rhyner, K.; Castagnola, J., et al. Role of transferrin, Fe, and transferrin receptors in myeloid leukemia cell growth. J. Clin. Invest. 75: 1061–1067; 1985.
Tsao, M. C.; Walthall, B. J.; Ham, R. G., Clonal growth of normal human epidermal keratinocytes in a defined medium. J. Cell. Physiol. 110: 219–229; 1982.
Wang, R. J.; Nixon, B. T. Identification of hydrogen peroxide as a photoproduct toxic to human cells in tissue culture medium irradiated with “daylight” fluorescent light In Vitro 14: 715–716; 1978.
Zigler, J. S., Jr.; Lepe-Zuniga, J. L.; Vistica, B., et al. Analysis of the cytotoxic effects of light-exposed HEPES-containing culture medium. In Vitro Cell. Dev. Biol. 21: 282–287; 1989.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Darfler, F.J. A protein-free medium for the growth of hybridomas and other cells of the immune system. In Vitro Cell Dev Biol 26, 769–778 (1990). https://doi.org/10.1007/BF02623618
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02623618