Obtaining a sufficient amount of healthy keratinocytes from a small tissue is difficult. However, ORS cells can be a good source of epithelium since they are easily obtainable and patients do not have to suffer from scar formation at donor sites. Accordingly, the current study modified the conventional primary culture technique to overcome the low propagation and easy aging of epithelial cells during culturing. In a conventional primary culture, the average yield of human ORS tells is 2.↑ $ imes$ 10$^3$cells/follicle based on direct incubation in a trypsin (0.1%)/EDTA(0.02%) solution for 15 min at 37$^{circ}C$, however, our modified method was able to obtain about 6.9 $ imes$ 10$^3$cel1s/follicle using a two-step enzyme digestion method involving dispase (1.2 U/mL) and a trypsin (0.1%)/EDTA (0.02%) solution. Thus, the yield of primary cultured ORS cells could be increasd three times higher. Furthermore, a total of 2.0 $ imes$ 10$^{7}$ cells was obtained in a serum-free medium. while a modified E-medium with mitomycin C-treated feeder tells produced a total of 6.3 $ imes$ 10$^{7}$ Cel1s over 17 days When Starting With 7.5 $ imes$ 10$^4$cells. Finally, We Confirmed the effectiveness of our ORS tell isolation method by presenting their ability for reconstructing the bioartificial skin epithelium in vitro