Development of novel intelligent tissue culture surfaces to accelerate cell proliferation and recover cell sheet under serum free conditions, using physiologically active peptides

为了发展先进的细胞片工程方法，我们开发了新型温度响应性聚合物接枝组织培养聚苯乙烯(TCPS)，将2-羧基异丙基丙烯酰胺单体与IPAAm单体进行共聚，将生理活性多肽固定在IPAAm单体上(PIPAAm-CIPAAm-TCPS)。用RGD多肽修饰的新型培养表面PIPAAm-CIPSSm-TCPS即使在无血清条件下也能培养细胞，并通过降温成功地将细胞从表面回收为片状。在这种新型表面上进行细胞培养，有助于避免细胞培养过程中由于无血清条件而造成的蛋白和病毒污染。RGD和PHSRN修饰的表面表现出比单独的RDG更好的细胞粘附性。相反，在新的培养表面引入RDG和胰岛素可以加速培养细胞的增殖。将生物素标记的RGD多肽引入到新型表面修饰的亲和素蛋白上。得到的RGD修饰表面即使在无血清条件下也表现出细胞黏附和增殖特性，这强烈表明含有生物素成分的各种生理活性多肽很容易固定在经亲和素修饰的新型表面上。确定合适的角膜上皮细胞、视网膜色素上皮细胞、肺泡细胞和心肌细胞的生理活性多肽，可以促进这类细胞膜的临床应用。

In order to evolve advanced method of cell sheet engineering, we have developed novel temperature responsive polymers grafted tissue culture polystyrene (TCPS), copolymerizing 2-carboxyisopropylacrylamide monomer as following immobilization of physiologically active peptides with IPAAm monomer (PIPAAm-CIPAAm-TCPS). The novel culture surfaces, PIPAAm-CIPSSm-TCPS, modified with RGD peptides attain culturing cells even under serum free conditions, and cells were successfully recovered as a sheet from the surfaces by lowering temperature. Cell culture onto the novel surfaces is useful for cells to avoid contamination of prion protein and virus during cell culture due to serum free condition. RGD and PHSRN modified surface exhibited more cell adhesive property than only RDG. In contrast, introduction of RDG and Insulin onto the novel culture surface achieve acceleration of proliferation of cultured cells. RGD peptides I inked with biotin were introduced onto avidin proteins modified onto the novel surfaces. Resulting RGD modified surfaces also showed cell adhesion and proliferation properties even under serum free conditions, strongly suggesting various physiologically active peptides, which contain biotin component, are easily immobilized onto the novel surfaces modified with avidin. Determination of appropriate physiologically active peptides for corneal epithelium cells, retinal pigment epithelium cells, alveolar cells and cardiac muscle cells may promote clinical application utilizing these kinds of cell sheets.