Summary
A survey of commercially availabla media revealed that medium F-12 was superior to medium 199 for clonal growth of human epidermal keratinocytes (HK) when supplemented with 10 μg/ml hydrocortisone (HC) plus dialyzed fetal bovine serum protein (FBSP), rather than the whole serum used in previous studies. Qualitative and quantitative adjustment of the medium composition for optimal clonal growth with minimal amounts of FBSP generated a new medium, MCDB 151, which supports clonal growth of HK with 10 μg/ml HC and as little as 1 mg/ml FBSP (equivalent in protein concentration to 2.0% whole serum). MCDB 151 differs significantly from MCDB 105, previously developed in this laboratory for normal human fibroblasts, and each medium selectively favors growth of its own type of cell in primary cultures of disaggregated human neonatal foreskin cells. Differences in the amounts of calcium and adenine in the two media appear to be among the most influential factors mediating the selective growth. Optimal growth of HK occurs at a very low level of Ca2+ that causes the colonies to remain as monolayers rather than stratifying as they do in the presence of higher levels of calcium. However, keratin synthesis, which was examined through use of highly specific fluorescent antibodies, is not affected by the Ca2+ concentration. Agents that increase intracellular cyclic AMP levels appear to have no effect on HK growth in MCDB 151 with 10 μg/ml HC and 1.0 mg/ml FBSP.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Peehl, D. M.; Ham, R. G. Growth and differentiation of human keratinocytes without a feeder layer or conditioned medium. In Vitro 16: 516–525; 1980.
Morgan, J. F.; Morton, H. J.; Parker, R. C. Nutrition of animal cell in tissue culture. I. Initial studies on a synthetic medium. Proc. Soc. Exp. Biol. Med. 73: 1–8; 1950.
Ham, R. G.; McKeehan, W. L. Nutritional requirements for clonal growth of nontransformed cells. Katsuta, H. ed. Nutritional requirements of cultured cells. Tokyo: Japan Scientific Societies Press; 1978: 63–115.
Ham, R. G. Clonal growth of mammalian cells in a chemically defined, synthetic medium. Proc. Natl. Acad. Sci. U.S.A. 53: 288–293; 1965.
McKeehan, W. L.; Genereux, D. P.; Ham, R. G. Assay and partial purification of factors from serum that control multiplication of human diploid fibroblasts. Biochem. Biophys. Res. Commun. 80: 1013–1021; 1978.
Rheinwald, J. G.; Green, H. Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells. Cell 6: 331–344; 1975.
McKeehan, W. L.; McKeehan, K. A.; Hammond, S. L.; Ham, R. G. Improved medium for clonal growth of human diploid fibroblasts at low concentrations of serum protein. In Vitro 13: 399–416; 1977.
McKeehan, W. L.; Ham, R. G. Stimulation of clonal growth of normal fibroblasts with substrata coated with basic polymers. J. Cell Biol. 71: 727–734; 1976.
Ham, R. G. Cloning of mammalian cells. Methods Cell Physiol. 5: 37–74; 1972.
McKeehan, W. L.; Hamilton, W. G.; Ham, R. G. Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblasts. Proc. Natl. Acad. Sci. U.S.A 73: 2023–2027; 1976.
Sun, T.-T.; Green, H. Keratin filaments of cultured human epidermal cells. Formation of intermolecular disulfide bonds during terminal differentiation. J. Biol. Chem. 253: 2053–2060; 1978.
Sun, T.-T.; Green, H. Immunofluorescent staining of keratin fibers in cultured cells. Cell 14: 469–476; 1978.
Liu, S.-C.; Karasek, M. Isolation and growth of adult human epidermal keratinocytes in cell culture. J. Invest. Dermatol. 71: 157–162; 1978.
Schneider, E. L.; Mitsui, Y.; Au, K. S.; Shorr, S. S. Tissue-specific differences in cultured human diploid fibroblasts. Exp. Cell Res. 108: 1–6; 1977.
Hennings, H.; Michael, D.; Cheng, C.; Steinert, P.; Holbrook, K.; Yuspa, S. H. Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 19: 245–254; 1980.
Gilbert, S. F.; Migeon, B. R.d-valine as a selective agent for normal human and rodent epithelial cells in culture. Cell 5: 11–17; 1975.
Green, H. Cyclic AMP in relation to proliferation of the epidermal cell: a new view. Cell 15: 801–811; 1978.
Ham, R. G.; McKeehan, W. L. Development of improved media and culture conditions for clonal growth of normal diploid cells. In Vitro 14: 11–22; 1978.
Puck, T. T.; Marcus, P. I. A rapid method for viable cell titration and clone production with HeLa cells in tissue culture: the use of X-irradiated cells to supply conditioning factors. Proc. Natl. Acad. Sci. U.S.A. 41: 432–437; 1955.
Ham, R. G. Nutritional requirements of primary cultures. A neglected problem of modern biology. In Vitro 10: 119–129; 1974.
Ham, R. G.; McKeehan, W. L. Media and growth requirements. Methods Enzymol. 58: 44–93; 1979.
Author information
Authors and Affiliations
Additional information
This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by Donna M. Peehl in partial fulfillment of the requirements for the Ph.D. degree.
This work was supported by Grant CA 15305 from the National Cancer Institute and Grant AG 00310 from the National Institute on Aging.
Rights and permissions
About this article
Cite this article
Peehl, D.M., Ham, R.G. Clonal growth of human keratinocytes with small amounts of dialyzed serum. In Vitro 16, 526–540 (1980). https://doi.org/10.1007/BF02626466
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02626466