An improved method for the isolation and culture of rat epidermal stem cells.
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.
Department of Burn Surgery, The First People's Hospital of Foshan Foshan, Guangdong, China.
Rong-Hua
RH
Yang
Ju-Lin
JL
Xie
Bin
B
Shu
Xu-Sheng
XS
Liu
Xiao-Dong
XD
Chen
Shu-Bin
SB
Ruan
Shao-Hai
SH
Qi
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002448:Cell Adhesion; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D000078404:Epidermal Cells; D004817:Epidermis / Q000196:embryology; D005865:Gestational Age; D019012:Integrin beta1 / Q000378:metabolism; D053569:Keratin-15 / Q000378:metabolism; D053539:Keratin-19 / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013234:Stem Cells / Q000378:metabolism / Q000502:physiology*; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D015415:Biomarkers; D019012:Integrin beta1; D053569:Keratin-15; D053539:Keratin-19; C108581:Tp63 protein, rat; D016159:Tumor Suppressor Protein p53
Epidermal stem cells; cell culture; fetal rat skin; stem cell isolation