Growth and ultrastructural characterization of proliferating human keratinocytes in vitro without added extrinsic factors

Routine in vitro cultivation of human epithelial cells derived from foreskin and free of contaminating fibroblasts has been achieved without the addition of conditioned medium or extrinsic factors. Epithelial cell populations could be serially subpassaged and exhibited modulating responses at PDL5 to culture conditions as the cells passed from phase 1 through phase 2 of their life span. The cell population in early phase 2 gave rise to tissue sheets that exhibited characteristics typical of human foreskin epidermis including the formation of distinct cellular layers, viz. strata basalis, spinosum, granulosum and corneum. Typical keratohyaline granules were not observed in the epithelial cells although a distinct cornified layer was evident. Ultrastructurally, desmosomes and tonofilaments were readily apparent. Thus, the procedure detailed in this study will produce highly differentiated fibroblast-free epidermal sheets reaching several centimeters in size and which can be removed from the substratum as a single sheet of organized epidermis. The epithelial cells could be cultured through 20±3 PDL, whereas fibroblast cultures derived from foreskin cultures exhibited 40±5 PDL and mixed cell cultures of foreskin were carried through 43±5 PDL.