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A new method to isolate human epidermal keratinocytes
Pages: 424-429
Year: Issue:  6
Journal: Journal of Clinical Dermatology

Keyword:  skinkeratinocytescell isolationcolony formation;
Abstract: Objective: To explore a new simple and efficient method to isolate human epidermal keratinocytes from tissues without separating the epidermis from the dermis. Methods: The skin tissue, chopped into small pieces by scalpel, was transferred to a digestion solution containing dispase and type I collagenase, and incubated at 37 ℃ water bath for 1 h, followed by incubation with Trypsin solution for 30 minutes, finished by incubation with DNase I for another 5 minutes. The digested solution was neutralized by adding same volume of DMEM containing 10% FBS, then filtered and centrifuged; The cell pellet was resuspended and plated with 10% FBS DMEM medium containing 10 mmol/L Rock inhibitor. 3 days later, the cells were fed with Cn T07 medium, and the medium was changed every other day. Results: Compared with the traditional dispase method, the new method isolated more keratinocytes; Cell growth and colony formation were faster; The passaged cells showed normal morphology and keratin 5 expression with a similar proliferative rate and the capability of colony formation to the cells isolated by traditional dispase method. Using the new method, epidermal keratinocytes were also successfully isolated from hairy scalp tissues, and the passaged cells expressed keratin 5. Conclusions: We established a new method to quickly and efficiently isolate high quality of epidermal keratinocytes. The new method can be used to isolate epidermal keratinocytes from different types of skin tissues including hairy scalp. This is a promising method for large-scale isolation of epidermal cells,which provides a crucial opportunity for cell-base therapy for skin injury.
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