Isolation And Culture Of Human Epidermal Stem Cells

Objective To research the methods for isolating,culturing and identifying human epidermal stem cells(ESC_S)and observe the growth charactericstics of ESCS in serum and serum-free medium. To carry on the exploration to a culture condition in order to harvest a good proliferation of the ESC_S in vitro. Methods â‘ keratinocytes and fibroblasts were separated from foreskin by Dispase â…¡and trypsin. We prepared the human fibroblast conditioned medium(HFCM) to epidermal stem cells medium(ESCM).The ESC_S isolated by rapidly adhering to collagen â…£were cultured with ESCM in vitro, taking the homologous keratinocytes as control. We observed the cell phenotypes and general growth condition, comparing CFE and CST with control. The expression of beta1 integrin and K₁₉ was identified with the immunocytochemical methods. â‘¡The ESC_S enriched as above were divided into two groups to culture and subculture in the serum and serum-free ESCM. The cellular form, CFE, growth curve and beta1 integrin and K19 strong positive expression rate were observed and compared in the two groups. Results â‘ The cells selected by rapid adherence to collagen â…£formed more colonies and reavealed the presence of holoclones after 2-week culture. In comparision with the unfractioned keratinocytes, the keratinocytes adhered more rapidly to type IV collagen had a significantly higher CFE which is 17.04±1.01% and 8.72±0.73%, respectively(P<0.01). Most of the isolated cells were strongly positive with immunocytochemical staining of K19 and beta1 integrin. â‘¡The cytomorphology analysis and the growth curve indicated that the ESCS in serum-free culture medium proliferated more obviously than those in serum culture medium during the 5-day cultivation, and achieved the cell basic confluence state at the 9th days, while the ESCS in serum medium continued to proliferate and climb the growth peak, entered the platform stage afterwards. The ESCS assumed the clone growth in two groups, but compared with the clone number and size, the CFE and CST in the serum group obviously surpassed that in the serum-free group(P<0.01). Cell cycle analysis demonstrated that G0^G1 stage cells proportion in the former was significantly higher than the latter(p<0.01). Strong positive expression percentage of beta1 integrin and K19 were significantly higher in ESCs cultivated in serum culture system than that of serum-free culture system(P<0.01). Conlusions The keratinocytes could be separated from foreskin by the Dispase II enzyme and trypsin, the ESCs could be isolated and enriched by rapid adherence to collagen â…£. The ESCM facilitated the ESCS culture in vitro. The charactericstics with the positive expression percentage of beta1 integrin and K19 and higher CFE during cell culture could be used for the identification of ESCS in vitro. Although both the serum ESCM and serum-free ESCM could be used for the ESCS culture, ESCS in the serum-free medium differentiated earlier, which was not favor of massive amplification in vitro. The cell phenotype maintenance and proliferation of the ESCS benefited from the serum ESCM in vitro.