Serum free medium for use in the formation of a histologically complete living human skin substitute

Methods and formulations are disclosed for the in vitro formation of a histologically complete human epidermis in a serum-free, companion cell or cell feeder layer-free, and organotypic matrix-free culture system commencing with the isolation and cultivation of a unique population of clonally-competent basal epidermal cells and ending with the formation of a functional, histologically complete, human squamous epithelium. The formation of a histologically complete human epidermis is accomplished in a serum-free medium, without companion-cells or feeder layer cells or any organotypic support using a multi-step process that is controlled by manipulating the growth and differentiation factors requisite to the sequential development of a usable, functional, and completely differentiated epidermis. The entire culture process can be accomplished in a relatively short time (3 to 4 weeks) with complete reproducibility and can supply copious amounts (2 to 3 square meters) of viable reformed human epidermis for a variety of experimental, clinical and commercial purposes where a histologically-complete living skin substitute is required.