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Hair follicle stem cells are vital seed cells for hair follicle and tissue engineering skin. However, there is no valid approach to obtain abundant cells within short times in vitro. The purpose of this study was to establish a new medium for culture and expansion of hair follicle outer root sheath (ORS) cells. ORS cells were harvested from human samples and cultured with a medium mixed with keratinocyte serum free medium and mouse embryonic fibroblasts suspension. The proliferation and vitality of ORS cells were evaluated. The cells exhibited typical cobblestone morphology with good adhesion and colony-forming ability. The cells were also characterized through detecting specific markers with immunofluorescent staining and the results showed that partial ORS cells could express hair follicle stem cell markers of CK15, CK19 and FST. At last, ORS cells and fibroblasts were used as seed cells and preliminarily constructed tissue engineering skin successfully. In conclusion, these findings suggested that this new medium could effectively accelerate cell proliferation and maintain the fundamental characteristics of ORS cells.