Temperature-Responsive Cell Culture Surfaces Enable “On−Off” Affinity Control between Cell Integrins and RGDS Ligands

Abstract

In this study, specific interactions between immobilized RGDS (Arg-Gly-Asp-Ser) cell adhesion peptides and cell integrin receptors located on cell membranes are controlled in vitro using stimuli-responsive polymer surface chemistry. Temperature-responsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) (P(IPAAm-co-CIPAAm)) copolymer grafted onto tissue culture grade polystyrene (TCPS) dishes permits RGDS immobilization. These surfaces facilitate the spreading of human umbilical vein endothelial cells (HUVECs) without serum depending on RGDS surface content at 37 degrees C (above the lower critical solution temperature, LCST, of the copolymer). Moreover, cells spread on RGDS-immobilized surfaces at 37 degrees C detach spontaneously by lowering culture temperature below the LCST as hydrated grafted copolymer chains dissociate immobilized RGDS from cell integrins. These cell lifting behaviors upon hydration are similar to results using soluble RGDS in culture as a competitive substitution for immobilized ligands. Binding of cell integrins to immobilized RGDS on cell culture substrates can be reversed spontaneously using mild environmental stimulation, such as temperature, without enzymatic or chemical treatment. These findings are important for control of specific interactions between proteins and cells, and subsequent "on-off" regulation of their function. Furthermore, the method allows serum-free cell culture and trypsin-free cell harvest, essentially removing mammalian-sourced components from the culture process.

Fabrication of Thermo-Responsive Molecular Layers from Self-Assembling Elastin-Like Oligopeptides Containing Cell-Binding Domain for Tissue Engineering

“…These wettabilities of the obtained oligo(ELP) layers were convenient for cell attachment, since the many cells are known to be hard to adhere to the hydrophilic surface [10][11][12][13]. …”

“…From these points of view, several types of peptide-containing block polymers have been developed as synthetic scaffolds for tissue engineering [6][7][8][9]. Thermo-responsive polymers with a lower critical solution temperature (LCST), such as poly(N-isopropylacrylamide) (PNIPAM), have also been used as functional surface especially in the field of cell-sheet engineering [10][11][12][13]. A pioneer work on cell-sheet engineering was reported by Okano and coworkers, who employed PNIPAM-grafted surface and accomplished the successful recovery of cultured cell as a sheet simply by temperature change [10][11][12][13].…”

“…Thermo-responsive polymers with a lower critical solution temperature (LCST), such as poly(N-isopropylacrylamide) (PNIPAM), have also been used as functional surface especially in the field of cell-sheet engineering [10][11][12][13]. A pioneer work on cell-sheet engineering was reported by Okano and coworkers, who employed PNIPAM-grafted surface and accomplished the successful recovery of cultured cell as a sheet simply by temperature change [10][11][12][13]. In this case, the PNIPAM-surfaces are hydrophobic at 37 °C and therefore allow cells adhesion, but the surfaces change to hydrophilic reversibly by lowering temperature below 32 °C and to be not cell adhesive due to rapid hydration of the grafted polymer.…”

Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

“…Moreover, functionalized substrates that capture or expose a cell-adhesive peptide on their surfaces in response to an external stimulus, such as heat [5], voltage [6][7][8] or light [9][10][11], are useful for the dynamic control of cell adhesion. These substrates, called dynamic substrates, have been applied not only to explore cell migration but also to coculture heterotypic cells [12].…”

Fabrication of a thermoresponsive cell culture dish: a key technology for cell sheet tissue engineering

“…carboxylate side chain, is copolymerized with IPAAm [56,57]. Covalent immobilization of the RGDS (Arg-Gly-Asp-Ser) peptide is performed through an amide bond onto a poly(IPAAm-co-CIPAAm)-grafted TCPS dish (figures 6(a) and (b)) [58,59]. Cells adhere and spread onto the RGDS-immobilized thermoresponsive TCPS in the absence of serum at 37…”