Long-term culture of murine epidermal keratinocytes

The production of transgenic and null mice with skin abnormalities makes it increasingly important to establish cultures of mouse epidermal keratinocy tes for in vitro studies. This requires that each cell line be derived from a single mouse and that the cells be carried for multiple passages. Freezi ng the cells would also be advantageous by allowing comparison of keratinoc ytes from several mouse lines at the same time. Mouse keratinocytes, howeve r, have been exceedingly difficult to grow as primary cultures, and subcult uring these cells has been virtually impossible until now. We describe a ge ntle dissociation method and a highly supplemented fibroblast conditioned m edium that allows us to grow and subculture total mouse keratinocytes for u p to 19 subcultures, allowing an increase in cell number of greater than 10 logs. Epidermal keratinocytes from newborn mice were grown on collagen PV coated dishes in murine fibroblast conditioned medium with 0.06 mM calcium and added growth factors. The cells could be passaged, frozen as viable sto cks, and induced to differentiate, Morphologically the cultured keratinocyt es demonstrated a pattern characteristic of basal cells. Stratified culture s which made mouse keratin 1 and profilaggrin through passage 10 were induc ed by purging the monolayer cultures of growth factors, then adding medium with 0.15 mM calcium; expression of mouse keratin 1 and profilaggrin was lo st by passage 15. The methods explained in detail here should be of great i nterest to investigators who are now trying to analyze skin phenotypes and expression of markers of epidermal differentiation of their transgenic or k nockout mice.