SEPARATION AND CULTURE OF EPIDERMAL STEM CELL in vitro
- VernacularTitle:表皮干细胞体外分离与培养
- Author:
Jianfu LI
;
Xiaobin FU
;
Zhiyoun SHENG
- Publication Type:Journal Article
- Keywords:
epidermal stem cells;
cell culture;
cell separation;
laboratory techniques and procedures;
in vitro
- From:
Medical Journal of Chinese People's Liberation Army
1983;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
To investigate the culture method for epidermal stem cell in vitro. Rat skin was peeled from the subcutaneous tissue gently, and cut into small pieces, then incubated for 24h in 0 25%trypsin in a 4℃ shaker. The epidermis was separated from the dermis, and shaken for 10min in 0 25% trypsin at 37℃ to dissociate into single cells. Digestion was terminated by the addition of culture medium +10% serum, and the cells were gently centrifuged and resuspended in the culture medium, which constituted EMEM without calcium, supplemented with 0 05mmol/L CaCl 2 , 9% chelexed FBS, 4ng/ml EGF, 0 5?g/ml gentamicin, and 50% fibroblast conditioned medium (CM ). The CM ( EMEM without calcium, 0 05mmol/L CaCl 2 , 9% chelexed FBS, 0 5?g/ml gentamicin ) was collected from freshly isolated primary neonate fibroblast cultures after 48h, which were passed through a filter with 0 45?m pores, and stored at -20℃ for use. 1?10 6 /ml epidermal cells were incubated for 10～15min at 37℃ on dishes coated with collagen Ⅳ, then the nonadherent cells were rinsed off. The adherent cells were grown to confluency. Cultures were subcultured in a solution of TGG for 5～10min. Once free of the dish, cells were centrifuged, resuspended in fresh medium, and then cultured at 1?10 5 /ml. Cultures were observed for colony formation under a phase contrast microscope, and the structure of the rapidly adherent cells was observed under electron microscopy; the expression of ? 1 integrin and K19 was detected in the rapidly adherent cells and colony cells with SP immunohistochemical methods. The results showed that the cells selected by rapid adherence to collagen type Ⅳ formed large colonies at 7 days, and showed a im mature feature under electron microscopy, manifesting ? 1 integrin and K19 expression. It is suggested that epidermal stem cells could be cultured in vitro .
