Abstract: Objective To study epidermal stem cell isolation and culture in vitro in order to provide experimental basis for tissue engineering of skin.Methods With heat-cool alternative method and trypsin digestion of tissuemass,the epidermal stem cells were cultured and observed continuously in vitro.The isolated cells were investigated on molecule β₁ integrin,K 15 and p63 by mimunohisto chemistry staining and scanning micro scope.Results The positive expressions of molecule β₁ in tegrin,K 15,and p63 and the spindle-and angular like cells in cluster growth were observed with scanning micro scope.The culture system was stable for the growth and classification of the stem cells and there existed affiliated organ of the skin in the culture system.Conclusion with the culture system developed,the epidermal stem cells isolated presents obvious biologic features and differentia tive trend with the formation of affiliated skin organ.