A flexible thermoresponsive cell culture substrate for direct transfer of keratinocyte cell sheets

Abstract

Most cell sheet engineering systems require a support or carrier to handle the harvested cell sheets. In this study, polyethylene terephthalate-based overhead projection transparency sheets (OHPS) were subjected to surface hydrolysis by alkali treatment to increase pliability and hydrophilicity and enable poly(N-isopropylacrylamide-co-glycidylmethacrylate) copolymer (NGMA) coating to impart thermoresponsiveness. NGMA was applied on the modified OHPS by the technique of spin coating using an indigenously designed spin coater. The spin coating had the advantage of using low volumes of the polymer and a reduced coating time. The surface chemistry and thermoresponsive coating was analyzed by Fourier transform infrared spectroscopy and water contact angle. Human keratinocyte cells were cultured on the spin coated surface and scaffold-free cell sheets were successfully harvested by simple variation of temperature. These cell sheets were found to be viable, exhibited epithelial characteristic and cell-cell contact as confirmed by positive immunostaining for ZO-1. The integrity and morphology of the cell sheet was confirmed by stereomicroscopy and E-SEM. These results highlight the potential of the NGMA spin coated modified OHPS to serve as a thermoresponsive culture surface-cum-flexible transfer tool.

Autogenous Biofabrication of Nativelike, Scaffold-Free Human Skin Equivalents Using a Smart, Enzyme-Degradable Tissue Templating Coating

“…More recently, cell sheet technology has been applied to generate dermal and epidermal tissues solely comprising cells bound together by their own adhesion molecules and extracellular matrix (ECM). , These cell sheets are generated by growing dermal fibroblasts or keratinocytes with a serum-supplemented medium (for enhanced cell and ECM density), using temperature-responsive substrates (i.e., surfaces that change their wettability with temperature) to “repel” cells and facilitate tissue detachment without the need for enzyme treatments. Tissues generated through this facilitated detachment method have shown an improved capacity to adhere to other tissues, allowing the scaling of their hierarchical organization (through stacking) and better graft integration after transplantation …”