Enhanced Proliferation and Functions ofIn VitroExpanded Human Hair Follicle Outer Root Sheath Cells by Low Oxygen Tension Culture

Abstract

Human hair follicle outer root sheath (hORS) cells are known to contain hair follicle stem cells and play an important role in healing large size wounds, and thus can serve as the cell source for skin engineering. This study investigated the effect of low oxygen tension culture on hORS cell proliferation potential and functional maintenance during in vitro expansion. Spared postsurgery scalp tissues were donated by 15 patients aged 20-45 (13 men and 2 women) and were randomly divided into three groups, and isolated hORS cells were combined into three pooled cell samples. They were cultured either in 4% O(2) or 21% O(2) and were analyzed for cell proliferation, colony forming efficiency (CFE), and their ability in forming engineered skin in vitro. The results showed that freshly isolated hORS cells expressed CD200 (22.88±8.76), cytokeratin 15 (CK15) (62.57±4.72), CD29 (22.53±2.49/strong and 29.80±4.09/dim), and CD49f (28.07±15.76/strong and 49.73±5.65/dim). When exposed in 4% O(2), hORS cells proliferated significantly faster than the cells in 21% O(2) for the first three passages (p<0.05), could better maintain cobblestone morphology, respectively, generate 3.63-folds more and 23.26-folds more cell yields after one and three passages. Additionally, enhanced CFE with significantly higher total and holoclone colony numbers were found in the 4% O(2) group than in the 21% O(2) group (p<0.05) for the first three passages along with better maintained CK15 expression. Furthermore, hORS cells expanded in 4% O(2) could form better epidermal structure of in vitro engineered skin comparing to the skin engineered by the control cells. The low oxygen culture method of hORS cells is simple, low cost, less labor intensive, and less biosafety concern, which may potentially be applied in skin engineering and clinical application.