It has been more than 30 years since the serial cultivation of human keratinocytes in monolayer culture was first described by Rheinwald and Green. Initially, isolation of primary keratinocytes from disaggregated human skin tissue and subsequent propagation was promoted through use of replication-inactivated murine fibroblast feeder layers. Since then numerous advances have been made to the cultivation of human keratinocytes in both two-dimensional monolayer and three-dimensional organotypic culture. Monolayer culture facilitates keratinocyte proliferation, whereas organotypic culturing techniques promote keratinocyte differentiation using conditions permissive for stratification. The protocols presented here describe traditional culturing methods, providing guidance for isolation and serial cultivation of primary human keratinocytes and dermal fibroblasts, as well as the use of these cells types for generation of stratified skin tissue.
