Quantitative analysis of cell responses based on dynamic properties of cell-adhesive molecules

Abstract

Event Abstract Back to Event Quantitative analysis of cell responses based on dynamic properties of cell-adhesive molecules Misaki Ito1, Yuuki Inoue2 and Kazuhiko Ishihara1, 2 1 The University of Tokyo, Department of Bioengineering, School of Engineering, Japan 2 The University of Tokyo, Department of Materials Engineering, School of Engineering, Japan Introduction: To controll cell responses is essential in regenerative medicine based on cell engineering. Cell responses are initiated from cell adhesion triggered by the interactions between the cell-adhesive proteins and corresponding receptors on cell membrane. Generally, the cell-adhesive proteins are part of the extracellular matrix with a different density, orientation and mobility. Therefore, controlling cell responses at the material sufraces, to understand an influence of the state of the proteins on cell adhesion is important. The purpose of this study is to analyze cell adhesion quantitatively from the viewpoint of the dynamic properties of cell-adhesive proteins. The non-specific interactions between proteins in cell membrane and that on material surfaces should be eliminated. Therefore, the cell-adhesion-motif-immobilized polymer brush layers composed of the poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) (PMPC) were constructed using surface-initiated atom transfer radical polymerization (ATRP). By controlling the three-dimensional structures of these polymer brush layers, the influence of dynamic properties of the cell-adhesive molecules can be elucidated. Experimental: The graft density of PMPC in polymer brush layer on gold substrate could be controlled from 0.01 to 0.18 chains/nm2 by the ratio of the ATRP-initiator group to the hydroxyl group. The change in the resonance frequency under dry condition (∆f) and in the dissipation under wet condition (∆D) by the formation of the PMPC brush layers were measured using the quartz crystal microbalance with dissipation (QCM-D) method. The amount of the adsorbed proteins from 10% fetal bovine serum was measured by QCM-D. As the cell adhesion motif, arginine-glycine-asparatic acid (RGD) was introduced at the terminal of the PMPC chains with the desired density. The HeLa cells were cultured on the prepared surfaces for 1 h and the number of the adherent cells was counted. Results and Discussion: The ∆D/∆f value, an index of the polymer chain mobility[1], decreased as the graft density increased. Thus, the mobility of immobilized RGD under wet condition could be controlled by the graft density of PMPC because the RGD was immobilized at the terminal of PMPC chains.The amount of the adsorbed proteins on the RGD-immobilized PMPC brush surfaces was quite low (less than 12 ng/cm2). Since the minimum value of the protein adsorption required for cell adhesion is 30 ng/cm2, these results indicated that the peptide-immobilized PMPC brush surfaces with different graft densities could induce cell adhesion via only the RGD. Number of adherent cells on the RGD-immobilized PMPC brush surfaces with the same RGD density increased as the ∆D/∆f value increased. From these results, we concluded that the high mobility of cell-adhesive molecules could promote cell adhesion.