Summary
Using gels of acid-soluble, collagen as a culture surface, trypsin-released keartinocytes from 0.1-mm, split-thickness sections of newborn foreskin may be plated with high efficiency and subcultured at a 1∶5 split a 2- to 3-week intervals for three subpassages. When plated at a density of 3.2×104 cells per cm2, keratinocytes attach to the gel with an efficiency of over 70%; after a lag phase of 3 days, the cells multiply exponentially with a doubling time of 60 hr. Cultures reach a growth-plateau phase at a density of 47.7×104 cells per cm2. Both hydrocortisone and epidermal growth factor (EGF) stimulate slightly the growth of primary cultures; both factors are required for proliferation of the 2nd and further passage of keratinocytes. As the cultures reach, confluence multilayers, of stratified cells are formed and cells of squamous morphology are spontaneously released from the surface. When the released cells and the attached cells are pulsed with [3H]-histidine and [14C]-leucine, a higher ratio of histidine to leucine is observed in the released cells indicating the biochemical onset of maturation. Orange G-Aniline Blue staining of the released cells show some of the cells to be completely keratinized. Fibrous proteins extracted from the cultured cells and analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis display the characteristic stratum corneum proteins of 60,000 and 66,000 daltons.
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Briggaman, R. A., D. C. Abele, S. R. Harris, and C. E. Wheeler. 1967. Preparation and characterization of a viable suspension of postembryonic human epidermal cells. J. Invest. Dermatol. 48: 159–168.
Karasek, M. A., and M. Charlton. 1971. Growth of postembryonic skin epithelial cells on collagen gel. J. Invest. Dermatol. 56: 205–210.
Yuspa, S. H., and C. C. Harris. 1974. Altered differentiation of mouse epidermal cells treated with retinyl acetate in vitro. Exp. Cell Res. 86: 95–105.
Elgjo, K., H. Hennings, D. Michael, and S. Yuspa. 1976. Natural synchrony of newborn mouse epidermal cells in vitro. J. Invest. Dermatol. 66: 292–296.
Fusenig, N. E., and P. K. M. Worst. 1974. Isolation and cultivation of epidermal cells from adult mouse skin. J. Invest. Dermatol. 63: 187–193.
Regnier, M., C. Delescluse, and M. Prunieras. 1973. Studies on guinea pig skin cell cultures. I. Separate cultures of keratinocytes and dermal fibroblasts. Acta Derm. Venereol., (Stockh.) 53: 241–247.
Cruickshank, C. N. D., J. R. Cooper, and C. Hooper. 1960. The cultivation of cells from adult epidermis. J. Invest. Dermatol. 34: 339–342.
Wheeler, C. E., C. M. Canby, and E. P. Cawley. 1957. Long-term tissue culture of epithelial-like cells from human skin. J. Invest. Dermatol. 29: 383–392.
Rheinwald, J. G., and H. Green. 1975. Serial cultivation of strains of human epidermal kerationocytes: the formation of keratinizing colonies from single cells. Cell 6: 331–341.
Rheinwald, J. G., and H. Green, 1977. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 265: 421–424.
Liu, S. C., and M. A. Karasek. 1978. Isolation and growth of adult human epidermal keratinocytes in cell culture. J. Invest. Dermatol. 71: 157–162.
Kissane, J., and E. Robins. 1958. The fluorometric measurement of deoxyribonucleic acid in animal tissue with special reference to the central nervous system. J. Biol. Chem. 233: 184–188.
Liu, S. C., and M. A. Karasek. 1978. Isolation and serial cultivation of rabbit epithelial cells. J. Invest. Dermatol. 70: 288–293.
Ayoub, P., and G. Shklar. 1963. A modification of the mallory connective tissue stain as a stain for keratin. Oral Pathol. 16: 580–581.
Steinert, P., and W. Idler. 1975. The polypeptide composition of bovine epithelial α-keratin. Biochem. J. 151: 603–614.
Bullaro, J. C., and D. H. Brockman. 1976. Comparison of skeletal muscle monoclayer cultures initiated with cells dissociated by vortex and trypsin methods. In Vitro 12: 564–570.
Laishes, B. A., and G. M. Williams. 1976. Conditions affecting primary cell cultures of functional adult rat hepatocytes. II. Dexamethasone enhanced longevity and maintenance of morphology. In Vitro 12: 821–832.
Baker, J. B., G. S. Barsh, D. H. Carney, and D. D. Cunningham. 1978. Dexamethasone modulates binding and action of epidermal growth factor in serum-free cell culture. Proc. Natl. Acad. Sci. U.S.A. 75: 1882–1886.
Cox, A. J., and E. P. Reaven. 1967. Histidine and keratohyalin granules. J. Invest. Dermatol. 49: 31–34.
Fukuyama, K., and W. L. Epstein. 1966. Epidermal keratinization: localization of isotopically labeled amino acids. J. Invest. Dermatol. 47: 551–560.
Steinert, P., and S. H. Yuspa. 1978. Biochemical evidence for keratinization by mouse epidermal cells in culture. Science 200: 1491–1493.
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Supported in part by Grants AM 14121 and AM 19595 of the U.S. Public Health Service.
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Liu, SC.C., Eaton, M.J. & Karasek, M.A. Growth characteristics of human epidermal kerationcytes from newborn foreskin in primary and serial cultures. In Vitro 15, 813–822 (1979). https://doi.org/10.1007/BF02618308
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DOI: https://doi.org/10.1007/BF02618308