Detachment of bovine corneal endothelial cell sheets by cooling-induced surface hydration of poly[(R)-3-hydroxybutyrate]-based thermoresponsive copolymer coating

Abstract

Cell sheet technology (CST) is a fascinating scaffoldless tissue engineering technique to generate a physiologically representative tissue replacement from autologous sources. As compared to conventional enzymatic cell harvesting methods, CST enables the preservation of important cell-to-cell junctions and extracellular matrix (ECM) components. However, covalent grafting methods are often employed for CST. In this study, a series of triblock copolymers with a hydrophobic and biocompatible poly[(R)-3-hydroxybutyrate] (PHB) central block flanked by varying lengths of terminal poly(N-isopropylacrylamide) (PNIPAAm) blocks (PNIPAAm-PHB-PNIPAAm) was synthesized via atom transfer radical polymerization of NIPAAm. The thermoresponsive triblock copolymers were explored as a non-covalent surface coating for culturing and detaching bovine corneal endothelial cell (BCEC) sheets. Aqueous solutions of the triblock copolymers produced thermosensitive micelles which can be drop-casted on glass substrates, resulting in a temperature-responsive surface. Importantly, incorporating a central hydrophobic PHB block enabled the anchoring of the coating to the bare substrate and enhanced the proliferation rate of the BCECs studied. Effective detachment of an intact cell sheet was also demonstrated via a cooling treatment at 4 °C for 20 min, and the viability of the detached cell sheet was found to be unaffected by the cooling. This work may potentially inspire more studies involving the non-covalent thermoresponsive polymer coatings for corneal tissue engineering applications.