Summary
A new medium (MCDB 104) has been developed which will support clonal growth of WI-38 cells at concentrations of serum protein as low as 25 μg per ml (equivalent to 0.05% serum). The principal factors responsible for reduction of the protein requirement are: (a) adjustment of all nutrient concentrations in medium F12 to experimentally determined optimum values for WI-38 cells; (b) supplementation with trace elements; (c) replacement of hypoxanthine and folic acid with adenine and folinic acid; and (d) coating of the culture surface with polylysine. Individually, many of these modifications exert only a small effect on cellular growth at reduced protein concentrations, but collectively their effect has been very substantial. Other strains of fibroblast-like human diploid cells from amniotic fluid, fetal lung and newborn foreskin also will grow at reduced concentrations of serum protein in the new medium.
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References
Ham, R. G. 1965. Clonal growth of mammalian cells in a chemically defined, synthetic medium. Proc. Natl. Acad. Sci. U.S.A. 53: 288–293.
Ham, R. G., S. L. Hammond, and L. L. Miller. 1977. Critical adjustment of cysteine and glutamine concentrations for improved clonal growth of WI-38 cells. In Vitro 13: 1–10.
McKeehan, W. L., W. G. Hamilton, and R. G. Ham. 1976. Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 73: 2023–2027.
McKeehan, W. L., and R. G. Ham. 1976. Stimulation of clonal growth of normal fibroblasts with substrata coated with basic polymers. J. Cell Biol. 71: 727–734.
Wang, R. J. 1976. Effect of room fluorescent light on the deterioration of tissue culture medium. In Vitro 12: 19–22.
Hayflick, L., and P. S. Moorhead. 1961. The serial cultivation of human diploid cell strains. Exp. Cell Res. 25: 585–621.
Hayflick, L. 1965. The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37: 614–636.
Eagle, H. 1955. Nutrition needs of mammalian cells in tissue culture. Science 122: 501–504.
Leibovitz, A. 1963. The growth and maintenance of tissue-cell cultures in free gas exchange with the atmosphere. Am. J. Hyg. 78: 173–180.
Ham, R. G. 1963. An improved nutrient solution for diploid Chinese hamster and human cell lines. Exp. Cell Res. 29: 515–526.
Ham, R. G. 1974. Nutritional requirements of primary cultures. A neglected problem of modern biology. In Vitro 10: 119–129.
Sanford, K. K. L. T. Dupree, and A. B. Covalesky. 1963. Biotin B12, and other vitamin requirements of a strain of mammalian cells grown in chemically defined medium. Exp. Cell Res. 31: 345–375.
Price, F. M., J. Rotherham, and V. J. Evans. 1970. Some aspects of the role of vitamin B12 in nucleic acid synthesis in a neoplastic C3H mouse cell strain. I. Growth studies. J. Natl. Cancer Inst. 44: 85–90.
Higuchi, K., and R. C. Robinson. 1973. Studies on the cultivation of mammalian cell lines in a serum-free, chemically defined medium. In Vitro 9: 114–121.
Vallotton, M., U. Hess-Sander and F. Leuthardt. 1965. Fixation spontanée de la biotine à une proteine dans le serum humain. Helv. Chim. Acta 48: 126–133.
Haggerty, D. F., and G. H. Sato. 1969. The requirement for biotin in mouse fibroblast L-cells cultured on serumless medium. Biochem. Biophys. Res. Commun. 34: 812–815.
Swim, H. E., and R. F. Parker. 1958. Vitamin requirements of uterine fibroblasts, strain U12-79; their replacement by related compounds. Arch. Biochem. Biophys. 78: 46–53.
Ham, R. G. 1963. Albumin replacement by fatty acids in clonal growth of mammalian cells. Science 140: 802–803.
Holley, R. W., J. H. Baldwin, and J. A. Kiernan. 1974. Control of growth of a tumor cell by linoleic acid. Proc. Natl. Acad. Sci. U.S.A. 71: 3976–3978.
Ham, R. G. 1964. Putrescine and related amines as growth factors for a mammalian cell line. Biochem. Biophys. Res. Commun. 14: 34–38.
Pohjanpelto, P., and A. Raina. 1972. Identification of a growth factor produced by human fibroblasts in vitro as putrescine. Nature [New Biol.] 235: 247–249.
Herzenberg, L. A., and R. A. Roosa. 1960. Nutritional requirements for growth of a mouse lymphoma in cell culture. Exp. Cell Res. 21: 430–438.
Ham, R. G. 1972. Cloning of mammalian cells. Methods Cell Physiol. 5: 37–74.
Balk, S. D., J. F. Whitfield, T. Youdale, and A. C. Braun. 1973. Roles of calcium, serum, plasma and folic acid in the control of proliferation of normal and Rous sarcoma virus-infected chicken fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 70: 675–679.
Whitfield, J. F., J. P. MacManus, R. H. Rixon, A. L. Boynton, T. Youdale, and S. Swierenga. 1976. The positive control of cell proliferation by the interplay of calcium ions and cyclic nucleotides. A review. In Vitro 12: 1–18.
Underwood, E. J. 1971.Trace Elements in Human and Animal Nutrition. 3rd Edition. Academic Press, New York.
Eagle, H. 1973. The effect of environmental pH on the growth of normal and malignant cells. J. Cell Physiol. 82:1–8.
Ham, R. G., and T. T. Puck. 1962. A regulated incubator controlling CO2 concentration, humidity and temperature for use in animal cell culture. Proc. Soc. Exp. Biol. Med. 111: 67–71.
Taylor, W. G., A. Richter, V. J. Evans, and K K. Sanford. 1974. Influence of oxygen and pH on plating efficiency and colony development of WI-38 and Vero cells. Exp. Cell Res. 86: 152–156.
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This work was supported by Grant No. AG00310 from the National Institute on Aging, and by Contract No. 223-74-1156 from the Bureau of Biologics, U.S. Food and Drug Administration.
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McKeehan, W.L., McKeehan, K.A., Hammond, S.L. et al. Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protein. In Vitro 13, 399–416 (1977). https://doi.org/10.1007/BF02615100
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DOI: https://doi.org/10.1007/BF02615100