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

Abstract

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 °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.

Precise Engineering of Growth Factor Presentation Using Extracellular Microenvironment-Mimicking Microfluidic Microparticles

“…The diameter of the GelMA-Hep cores could be readily tuned by controlling the flow rate ratio of the polymer-containing aqueous phase to the oil phase (volumetric flow ratio; Figure b). Heparin is known to have a high affinity for various growth factors, including but not limited to FGF, VEGF, and BMPs. − To fine-tune the affinity of GelMA-Hep microparticles, we designed the system such that the heparinization degree can be easily controlled by adjusting the GelMA-to-heparin feeding ratio (Figure c). To demonstrate the high growth factor affinity of our GelMA-Hep cores, we utilized Bio-Layer Interferometry (BLI) sensorgrams to visualize the association/disassociation processes of rhBMP-2 to surfaces coated with GelMA, GelMA-Hep, or Heparin (Figure d).…”