Isolation，culture and identification of human skin epidermal stem cellexosomes / 安徽医科大学学报

ABSTRACT

Objective@#To explore method for isolating and culturing human epidermal stem cells ( EPSCs) in vitro and isolating and purifying epidermal stem cell exsomes ( EPSCs-Exo) by optimizing the technical process．@*Method@#Firstly，the improved separating enzyme was used to isolate the EPSCs derived from human skin tissue．Then，an improved serum-free culture medium and 10 specific factors were combined to construct optimized 2D culture medium which could stimulate the growth of EPSCs，promote the secretion of EPSCs-Exo，maintain the stemness and proliferation of EPSCs，and delay the differentiation and maturation of EPSCs． Further，the conditions of differential centrifugation was optimized，and then the human EPSCs-Exo was successfully extracted with high efficiency and high purity．@*Results@#The human skin tissue was confirmed with the expressions of markers for epidermal cells． EPSCs were verified with high expression levels of integrin-α6，integrin-β1，P63 and CK19 by immunofluorescence staining and Western blot． The nanoparticle tracking analysis results showed the particles separated for EPSCs supernatant was saucepan with the detected diameter between 30 － 150 nm． The Western blot results showed the positive expression of membrane markers Tsg101，CD9 and CD63 and the negative expression of intracellular markers Calnexin and GAPDH．@*Conclusion@#The results show that the human-derived EPSCs have been successfully isolated and cultured in vitro，and the EPSCs-Exo have been successfully isolated and identified．