Concomitant proliferation and formation of a stratified epithelial sheet by explant outgrowth of epidermal keratinocytes from adult mice

A chemically defined medium containing 1.2 mM Ca²⁺ has been developed for the culture of primary epidermal keratinocytes from untreated adult mice such that proliferation is accompanied by the formation of desmosomes and stratification. Cultured cutaneous explants of 1 mm² from the backs of untreated, control, and carcinogen-exposed mice all demonstrated epithelial outgrowth within 1 wk, and by 5 wk approached confluence with characteristics of terminal differentiation such as desmosomes and stratification. Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) to the medium in concentrations of 0.001, 0.01, and 0.1 μg/ml resulted in a delay of approximately 1 wk in the outgrowth of the explants compared with the acetone controls and in a 30% decrease in the diameter of the epithelial outgrowth at 3 wk. The inhibition in outgrowth was overcome at higher concentrations (0.5, 1.0, and 10 μg/ml TPA). No obvious differences in morphology or in the rate of epidermal outgrowth within a 5-wk interval among explants from normal untreated epidermis, epidermis from mice treated with acetone, or epidermis from mice treated with an initiating application of 7,12-dimethylbenza]anthracene were observed. The defined composition of this medium and its ability to support reproducibly and conveniently both proliferation and differentiation of normal as well as treated primary adult murine epidermal cells suggest that it should be useful for a number of studies not previously possible that are relevent to the biology of the skin, to toxicology, and to carcinogenesis in the murine model system.