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Hair follicle is a small but very complex and dynamic miniorgan of the human body. It is easy to isolate and culture mesenchymal cells but not epithelial cells of hair follicle. It is necessary for intact and healthy outer root sheath (ORS) cells to be isolated and cultured. In this study we developed an appropriate isolation method to yield 6.4 ± 0.75 × 104 cells/hair follicle, which is about 9-fold comparing to our previous data. This yield was achieved by modifications such as different kinds of enzyme uses, fragmentation, and mechanical stimuli. Especially we detected that the different kinds of isolation enzyme could affect proliferation of ORS cells during primary culture. In addition, bovine pituitary extract (BPE) was needed for ORS cells to proliferate and to form colonies under serum-free, feeder layer-free culture condition, but type I collagen as a substratum did not have any positive effect. Moreover, ORS cells under BPE-added condition contained stem/progenitor cells expressing β1-integrin, CK19, and CD34. These results can provide useful cell culture information, not only in the study of hair biology but also in the field of tissue engineering and cell therapy for the treatment of alopecia.