Phenotypical characterization of 6-21-week gestational age human dermis and epidermal cell isolation methods for in vitro studies on epidermal progenitors.

Cell banked epidermal skin progenitor cells have the potential to provide an “off-the-freezer” product. Such cells may provide a skin donor area-independent cell-spray grafting therapy for the treatment of burns. We first characterized fetal skin samples of gestational ages ranging from 6 to 21 weeks. As the results suggest that the phenotypic differentiation occurs after 10 weeks, which may complicate follow-up in vitro studies, we developed and compared different cell isolation techniques for human fetal skin-derived epithelial cells from tissue ages 6 to 9 weeks. We initially screened seven methods of characterization, concluding that two methods warranted further investigation: incubating the epidermal tissue in Petri-dishes with culture medium for spontaneous cell outgrowth, and wiping the epidermal tissue onto a dry Petri-dish culture surface followed by adding culture medium. Non-controllable culture contamination with dermal cells was the reason for excluding the other five methods. The results suggest that epidermal cells can be isolated from tissue exhibiting a single homogeneous layer of CK15+ basal keratinocytes up to week 9. At later gestational ages, the ongoing skin differentiation results in a multi-layer basal structure and progenitors associated with the hair bulb would have to be considered. Spraying the resulting cells with a clinical spray device was successfully demonstrated in an in vitro model.