Terminus-Selective Covalent Immobilization of Heparin on a Thermoresponsive Surface Using Click Chemistry for Efficient Binding of Basic Fibroblast Growth Factor

Cell therapy using endothelial cells (ECs) has great potential for the treatment of congenital disorders, such as hemophilia A. Cell sheet technology utilizing a thermoresponsive culture dish is a promising approach to efficiently transplant donor cells. In this study, a new method to prepare terminus-selective heparin-immobilized thermoresponsive culture surfaces is developed to facilitate the preparation of EC sheets. Alkynes are introduced to the reducing terminus of heparin via reductive amination. Cu-catalyzed azide-alkyne cycloaddition (CuAAC) facilitates efficient immobilization of the terminus of heparin on a thermoresponsive surface, resulting in a higher amount of immobilized heparin while preserving its function. Heparin-immobilized thermoresponsive surfaces prepared using CuAAC exhibit good adhesion to human endothelial colony-forming cells (ECFCs). In addition, upon further binding to basic fibroblast growth factor (bFGF) on heparin-immobilized surfaces, increased proliferation of ECFCs on the surface is observed. The confluent ECFC monolayer cultured on bFGF-bound heparin-immobilized thermoresponsive surfaces exhibits relatively high fibronectin accumulation and cell number and detaches at 22 degrees C while maintaining the sheet-like structure. Because heparin has an affinity for several types of bioactive molecules, the proposed method can be applied to facilitate efficient cultures and sheet formations of various cell types. Click chemistry between thermoresponsive culture surfaces modified with azide groups and heparin with an alkynylated reducing terminus enables the efficient immobilization of functional heparin on the surface. This surface can hold bFGF efficiently, resulting in enhanced proliferation of endothelial cells (ECs). Therefore, EC sheets can be fabricated on bFGF-bound heparin-immobilized surfaces.image