Cell
Seria cultivation of strains of human epidemal keratinocytes: the formation keratinizin colonies from single cell is
Summary
Human diploid epidermal cells have been successfully grown in serial culture. To initiate colony formarion, they require the presence of fibroblasts, but proiiferation of fibroblasts must be controlled so that the epidermal cell population is not overgrown. Both conditions can be achieved by the use of lethally irradiated 3T3 cells at the correct denairy. When trypsinized human skin cells are plated together with the 3T3 cells, the growth of the human fibroblasts is largety suppressed, but the epidermal cels grow from singte ceils into cotonies. Each colony consists of keratinocytes ultimerely forming a stratified squamous epithelium in which the dividing cells are confined to the lowest layer(s). Hydrocortisone is added to the medium, since in secondary and subsequent subcultures it makes the coiony morphology more orderly and distinctive, and maintains proliferation at a slightly greater rate. Under these culture conditions, it is possible to isotate keratinocyte clones free of viable fibroblasts.
Like human diploid fibroblasts, human diploid keratinocytes appear fo have a finite caiture lifetime. For 7 strains studied, the culture lifetime ranged from 20–50 cell generations. The plating efficiency of the epidermal cells taken directly from skin was usually 0.1–1.0%. On subsequent transfer of the cultures initiated from newborns, the plating efficiency rose to 10% or higher, but was most often in the range of 1–5% and dropped sharply toward the end of their culture life. The plating efficiency and culture lifetime were lower for keratinocytes of older persons.
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