Temperature-responsive cell culture surfaces enable "on-off" affinity control between cell integrins and RGDS ligands.
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.
Department of Applied Chemistry, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan.
Mitsuhiro
M
Ebara
Masayuki
M
Yamato
Takao
T
Aoyagi
Akihiko
A
Kikuchi
Kiyotaka
K
Sakai
Teruo
T
Okano
D002448:Cell Adhesion / Q000502:physiology; D002452:Cell Count / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
D016023:Integrins; D008024:Ligands; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid