Separation and culture of human epidermal stem cells in vitro
Hu, K.; Dai, Y.; Li, J.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 20(12): 1244-1247
2006
ISSN/ISBN: 1002-1892 PMID: 17228692 Document Number: 596146
To investigate the culture method for epidermal stem cells in vitro. The epidermis was separated from the dermis, and shaken for 10 min in 0.05% trypsin at 37 C to dissociate into single cells. Epidermal stem cells were selected by rapid attachment to collagen IV for 10-15 min and cultured on collagen IV or 3T3 feeder layer. All the cells were grown in DMEM without calcium, supplemented with 10% chelexed fetal bovine serum, 10 microg/L epidermal growth factor, 0.05 mmol/L CaCl2 and 0. 8 mg/L hydrocortisone. Cultures were observed for colony formation under a phase contrast microscope. The phenotypes of epidermal stem cells were detected by flow cytometry and immunocytochemistry staining. The cells selected by rapid adherence to collagen IV formed large colonies at 7- 8 days, expressed K19 antigen. The percentages of cells at the G0 and G1 phases of the cell cycle and the percentage of alpha 6 briCD71 dim cells in the experimental groups were higher than those in the control group. It indicated that there was a significant difference between the experimental groups and the control groups(P<0. 05). The human epidermal stem cells can be selected by rapid attachment to collagen IV, and they can be expanded in culture if the appropriate conditions are maintained.