ABSTRACT
Human keratinocytes have been cultivated in vitro using different methods. Using small size skin, large epidermal sheets have been produced. These epidermal sheets have been used for the treatment of injured epidermis, especially burn and other skin clinical disorders, and also for research purposes. The objective of this study was isolation and in vitro cultivation of human skin keratinocytes and preparation of epidermal sheet without using mouse 3T3 fibroblast feeder layer. Small size human skin with a relative thickness was divided to small pieces and the epidermis was separated from dermis using warm trypsin. Then keratinocytes were isolated from epidermis and cultured with high density in DMEM containing FBS and other supplements like cholera toxin and epidermal growth factor. The resulting epidermal sheet was separated using dispase grade II and transferred to vaseline gauze. Histological studies were also carried out on the epidermal sheet. Keratinocytes grew as multiple colonies and produced a thin confluent epidermal sheet consisted of a few layers of cells. With time, number of cell layers was increased and a thicker epidermal sheet was produced. Fibroblasts, which were present in the original cell suspension, grew in the culture when the growth of keratinocyte was slow or when a low density of keratinocytes was used. The histological examination of epidermal sheet showed the presence of germinative basal cells, suprabasal cells with relative differentiation and also melanocytes. The dimension of epidermal sheet was decreased when it was separated from tissue culture flask using dispase. Cultured epidermal autografts have several clinical and investigative indications. Even though the cultivation of keratinocytes with high density is easy, however, the surface area of resulting epidermal sheet is limited and its subculture is difficult. In this method there is the risk of growth of dermal fibroblasts. To produce thicker epidermal sheet with more cellular layers, it is better to separate it within two to three weeks after cultivation