Immobilization of biomolecules onto thermoresponsive culture dishes by affinity binding

This paper describes a novel method to immobilize biomolecules such as growth factors and enzymes onto the surface of poly (N-isopropylacrylamide-co-2-carboxyisopropyl-acrylamide (poly(IPAAm-co-CIPAAm))-grafted tissue culture polystyrene dish (TCPS) via affinity binding between avidin and biotin. Effects of Arg-Gly-Asp-Ser (RGDS) peptides with different spacer length on the behavior of cell attachment/detachment were also examined. In cell attachment assay, human umbilical vein endothelial cells (HUVECs) adhered well on G₁₂RGDS-immobilized thermoresponsive TCPS at 37degC. However, weak adhesion of HUVECs was observed on thermoresponsive TCPS with shorter and longer peptides than G₁₂ RGDS. In cell detachment assay, adhered HUVECs were detached after lowering temperature from 37degC to 20degC or adding free RGDS solutions. Then the rate of cell detachment was accelerated by both lowering temperature and adding free RGDS solutions. Adding free RGDS solutions or changing surface density of RGDS peptides could also control the rate of HUVECs detachment from thermoresponsive TCPS. Consequently, thermoresponsive TCPS with G₁₂RGDS peptide allowed cell culture under the serum-free medium and noninvasive cell recovery by lowering temperature