SEPARATION AND CULTURE OF EPIDERMAL STEM CELL in vitro / 解放军医学杂志

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 .