Development of isolation and cultivation method for outer root sheath cells from human hair follicle and construction of bioartificial skin

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 cells is 2.1 × 10³ cells/follicle based on direct incubation in a trypsin (0.1%)/EDTA (0.02%) solution for 15 min at 37°C, however, our modified method was able to obtain about 6.9 × 10³ cells/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 × 10⁷ cells was obtained in a serum-free medium, while a modified E-medium with mitomycin C-treated feeder cells produced a total of 6.3 × 10⁷ cells over 17 days when starting with 7.5 × 10 ⁷ cells. Finally, we confirmed the effectiveness of our ORS cell isolation method by presenting their ability for reconstructing the bioartificial skin epithelium in vitro.