【Abstract】 Objective To search for a feasibil ity of repairing full-thickness cutaneous deficiency with tissueengineered skin substitute composited by human epidermal stem cells and fibroblasts in fibrin frame. Methods Epidermal stem cells and fibroblasts were harvested from human epidermis and dermis by trypsin digestion. Cells were cultured and subcultured in non-serum medium. Epidermal stem cells (5×104/mL) and dermal fibroblasts (1×104/mL) in 0.5 mL medium were coagulated in 0.5 mL fibrin frame to construct tissue engineered skin substitute. The tissue engineered skin substitute was grafted onto full-thickness cutaneous deficiency of nude mice. Forty-five male mice, 4-5 week old, weighted 20 g on average, were randomly divided into 5 groups. Oil yarn (group C), fibrin frame membrane without cell inoculation (group F), composite skin substitute with epidermal stem cells (group S) and composite skin substitute with fibroblasts (group Fb) were used as controls, while tissue engineered skin substitute (group T) was experimental group. The wounds were observed 1, 3, 6, 8 weeks after surgery. Samples were harvested 3, 6, 8 weeks after surgery, and were examined by means of histology、immunohistochemistryand scanning electron microscopy (SEM). Results Four weeks after cell culture, there were some round cells in the culture capsule of epidemic cells, and some fusiform cells in the culture capsule of fibroblast. Six days after cells were cultured in the BrdU culture medium, there were some BrdU positive cells appeared. There were some CK19 positive cells and Nestin positive cells appeared in the chaff of group T before transplanting. The new formed skin of group T grew faster and had less scar than other groups. Six weeks after surgery, the average thickness of new formed skin was (0.460 ± 0.049) mm in group C, (0.480 ± 0.055) mm in group F, (0.540 ± 0.043) mm in group S, (0.510 ± 0.032) mm in group Fb, (0.660 ± 0.047) mm in group T. The thickness of new formed skin in group T was thicker than other groups (P lt; 0.05). By histology and SEM observation, 3, 6, 8weeks after surgery, the new formed cuticular layer, fibroblast and blood vessels in the group T were more than those in theother groups. The al ignment of blood vessels and collagen fibers in group T were much regular than those in the other groups. Three weeks after surgery, the new formed skin of group T had a continuous color zone of positive collagen Ⅳ staining, while no continuous color zone was found in the other groups. Six weeks after surgery, CK14 positive cells appeared in the new formed skin of group T, while no positive cell was found in the other groups. Conclusion Tissue engineered skin substitute which is composited with epidermal stem cells and fibroblasts in fibrin frame has potential prospects in appl ication of repairing fullthickness cutaneous deficiency with advantage of faster wound heal ing.