[Separation and culture of human epidermal stem cells in vitro].
OBJECTIVE
To investigate the culture method for epidermal stem cells in vitro.


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Hu
Kuikui
K
Center of Plastic, Laser and Cosmetic Surgery, Second Affiliated Hospital of Jiangxi Medical Collage, Nanchang Jiangxi , 330006, P R China.
Dai
Yucheng
Y
Li
Jian
J
D000183:Actihaemyl; D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002455:Cell Division; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D000078404:Epidermal Cells; D006801:Humans; D013234:Stem Cells / Q000166:cytology*
D000183:Actihaemyl