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Int J Clin Exp Pathol 2013;6(11):2529-2534

Original Article
An improved method for the isolation and culture of rat epidermal stem cells

Rong-Hua Yang, Ju-Lin Xie, Bin Shu, Xu-Sheng Liu, Xiao-Dong Chen, Shu-Bin Ruan, Shao-Hai Qi

Department of Burn Surgery, The First People’s Hospital of Foshan, Foshan, Guangdong, China; Department of Burn Surgery, First Affiliated
Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, China. Equal contributors.

Received August 29, 2013; Accepted October 3, 2013; Epub October 15, 2013; Published November 1, 2013

Abstract: The management of burns and injuries using novel treatment strategies involving epidermal stem cells (ESC) requires a better
understanding of the biology of these cells, in particular, their isolation and the maintenance of their unique characteristics in culture. The
purpose of this study was to describe an improved method for isolating putative ESC from fetal rat skin and to maintain them long term in
culture. Single ESC suspensions were obtained from fetal rat skin by enzyme digestion containing 0.5% neutral protease. The target cells were
harvested by rapid adherence on type IV collagen plates and were cultured in complex DMEM. After primary isolation, cells were continuously
cultured in K-serum free medium. After reaching 70-80% confluence, the cells were digested with 0.25% trypsin at 37°C for 5-10 minutes, and
passaged at a ratio of 1:2. The cultured ESC showed good growth, resulting in cell viability of over 98%. Four days later, clones containing 100-
200 cells were detected, showing cobblestone-like characteristics. The rapidly adherent cells were positive for keratin 15, 19 and P63. Eighty
three percent of cells expressed β1 integrin. The growth-curve showed that the rapidly adherent cells were in the exponential growth phase.
The protocol described in this paper provides a simplified and effective method to isolate and maintain long-term culture of epidermal stem
cells from fetal rat skin. This method should be valuable for isolating and studying ESC from various transgenic rat lines that are currently
available. (IJCEP1308069).

Keywords: Epidermal stem cells, stem cell isolation, cell culture, fetal rat skin

Address correspondence to: Dr. Shaohai Qi, Department of Burns, First Affiliated Hospital, Sun Yat-Sen University, 58, 2nd Zhongshan Road,
Guangzhou, Guangdong, 510080, China. Tel: 0086-20-87335577; E-mail: a_hwa991316@hotmail.com