Harvesting epithelial keratinocyte sheets from temperature-responsive dishes preserves basement membrane proteins and improves cell survival in a skin defect model.

Cultured epithelial autograft (CEA) therapy has been used in clinical applications since the 1980s. However, there are some issues related to this treatment that still remain unsolved. Enzymatic treatment is typically used in the collection of epithelial keratinocyte sheets, but it tends to break the adhesion and basement membrane proteins. It is thought that the loss of proteins after enzymatic treatment is responsible for the poor survival of transplanted cell sheets. Our laboratory has developed a temperature-responsive culture dish that does not require enzymatic treatment to harvest the cells. In this study, we compare morphological and survival results from rat epithelial keratinocyte cell sheets harvested by temperature-reducing treatment (TT sheets) against cell sheets harvested by enzymatic (dispase) treatment (DT sheets). TT sheets preserve keratin structure in better conditions and express higher levels of collagen IV and laminin 5 than DT sheets. In order to evaluate cell sheet survival after transplantation, we created an in vivo transplant model. Keratinocyte sheets obtained from GFP-positive animals were transplanted into athymic rats. The survival rate 7 days after transplantation of TT sheet was higher than that of DT sheets. Collagen IV and Laminin 5 expression was observed in the TT sheet transplantation group. These results indicate that the remaining basement membrane proteins are important for initial attachment and cell survival. We believe that the cell sheet harvesting method using temperature-responsive culture dishes provides superior cell survival and can solve one of the roadblocks in CEA therapy. Copyright © 2016 John Wiley & Sons, Ltd.