Temperature-responsive cell culture surfaces enable "on-off" affinity control between cell integrins and RGDS ligands
Temperature-responsive cell culture surfaces enable "on-off" affinity control between cell integrins and RGDS ligands
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DOI：
10.1021/bm0343601
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发表时间：
2004-03-01
影响因子：
6.2
通讯作者：
Okano, T
中科院分区：
文献类型：
--
作者：
Ebara, M;Yamato, M;Okano, T
在这项研究中，固定RGDS（精氨酸-甘氨酸-天冬氨酸-丝氨酸）细胞粘附肽和细胞膜上的细胞整合素受体之间的特定相互作用的控制在体外使用刺激响应聚合物表面化学。接枝到组织培养级聚苯乙烯（TCPS）培养皿上的温度响应性聚（N-异丙基丙烯酰胺-co-2-羧基异丙基丙烯酰胺）（P（IPAAm-co-CIPAAm））共聚物允许RGDS固定化。这些表面促进了人脐静脉内皮细胞（HUVEC）在37 ℃（高于共聚物的低临界溶液温度，LCST）下无血清的铺展，这取决于RGDS表面含量。此外，在37 ℃下，通过将培养温度降低到低于LCST，细胞在RGDS固定的表面上铺展，从而自发地分离，因为水合接枝共聚物链使固定的RGDS与细胞整联蛋白解离。水合后的这些细胞提升行为类似于在培养物中使用可溶性RGDS作为固定化配体的竞争性取代的结果。细胞整联蛋白与细胞培养基质上固定的RGDS的结合可以使用温和的环境刺激（例如温度）自发逆转，而无需酶或化学处理。这些发现对于控制蛋白质和细胞之间的特异性相互作用以及随后对其功能的“开-关”调节非常重要。此外，该方法允许无血清细胞培养和无胰蛋白酶细胞收获，基本上从培养过程中去除了来自酵母的组分。
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 37degreesC (above the lower critical solution temperature, LCST, of the copolymer). Moreover, cells spread on RGDS-immobilized surfaces at 37degreesC 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.