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

Abstract

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.

Rapid fabrication of detachable three‐dimensional tissues by layering of cell sheets with heating centrifuge

“…The cell sheets maintain cell‐to‐cell junctions and extracellular matrix (ECM) and can be layered on temperature‐responsive culture dishes or normal polystyrene culture dishes for fabricating thicker tissues. Cell sheet technology has been applied to regenerating various injured tissues . Cell sheet‐tissues have been used in various clinical trials for damaged tissue regeneration …”

Influence of fibrin matrices and their released factors on epidermal substitute phenotype and engraftment

“…However, this positive effect on basement membrane production was not sufficient to reach the expression observed with hPBM or FPF scaffolds. Other studies have shown that the conservation of basement membrane before grafting was essential for a good graft take of epidermal substitutes (Osada et al, ). In our study, although the basement membrane of epidermal substitutes grown on hPBM was not fully organized before grafting (Data SS2), it may have contributed to a favorable effect on engraftment and DEJ development later on in vivo (Figure c).…”

“…Moreover, both FPF and hPBM are biodegradable materials that are removed physiologically after grafting by the fibrinolytic system (Janmey, Winer, & Weisel, 2009). Additionally, the detachment procedure of these two matrices (Alexaline et al, 2015;Meana et al, 1998) do not necessitate enzymatic treatment (Osada, Sekine, Soejima, Sakurai, & Shimizu, 2016), thus allowing the preservation of DEJ proteins. At last, rete ridges-like structures were observed 1 month after grafting epidermal substitutes grown on FPF but not CEA (Ronfard et al, 2000).…”

Keratinocyte sheets prepared with temperature-responsive dishes show enhanced survival after in vivo grafting on acellular dermal matrices in a rat model of staged bi-layered skin reconstruction

“…To our knowledge, Osada et al. are the only group to create epithelial KSs using TR culture, and to demonstrate that this approach improves KSs survival in vivo in a rodent model [21]. Here, we leveraged the experiences of Osada et al.…”

“…Osada et al. reported that KSs prepared with this method show better-preserved BMs and higher survival when grafted directly on wounds in a rat model [21], [22]. Application of this strategy to staged bi-layered skin reconstruction – a more common and relevant clinical scenario – has not been reported yet.…”