對於再生醫療與新藥研發而言，人類多能性幹細胞是具有前瞻性的細胞來源，為了避免於臨床應用造成異種汙染，開發化學定義成分的生醫材料來培養人類多能性幹細胞是重要的課題。有幾種生醫材料被發展來培養人類多能性幹細胞並維持其細胞多能性，例如: 塗佈重組玻連蛋白的培養盤以及嫁接從細胞外基質得來的寡肽培養盤。本研究探討合成材料的軟硬度如何影響細胞的繁殖和多能性。 本研究製作嫁接有重組玻連蛋白的高分子水凝膠(polyvinylalcohol-co-itaconic acid)薄膜來研究軟硬度的物理性質如何影響人類胚胎幹細胞 (WA09)的多能性及生長速率。藉由戊二醛交聯劑，我們可以調整高分子水凝膠的軟硬度至10.3 到 30.4 kPa 儲存模數，接著嫁接高分子賴氨酸在水凝膠上作為主鏈並調整表面電荷，最後嫁接重組玻連蛋白於基材上。 本研究調適出最優化的高分子水凝膠軟硬度並嫁接較低濃度的 (5 g/ml)重組玻連蛋白，相較於其他研究需要極高濃度的 (500 g/ml)寡肽。此材料被用來長期培養繁殖人類多能性幹細胞至少10到20代於無異種環境，並藉由螢光染色、胚體的體外分化和畸形瘤的體內生成來鑑定培養10到20代過後的細胞蛋白質是否具有多能性。 關鍵字： 多能性幹細胞、無異種、表面電荷、水凝膠、玻連蛋白 ;Abstract Human pluripotent stem cells (hPSCs) are promising cell source for regenerative medicine and drug discovery. The development of chemically defined biomaterials is necessary for culturing hPSCs for clinical applications without xenogenic contaminants. It is developed that the biomaterials for hPSCs culture to maintain their pluripotency, such as (a) dishes coated with recombinant vitronectin (b) dishes immobilized with oligopeptides derived from extracellular matrices (ECMs). It is investigated that the effect of the elasticity of the synthetic dishes on the pluripotency fate and proliferation of hPSCs in this study. I developed polyvinylalcohol-co-itaconic acid (PVA-IA) films grafted with recombinant human vitronectin (rVN) to evaluate the physical effect of elasticity of hydrogels grafted with biologically active nanosegments on the pluripotency and proliferation fates of hPSCs (WA09). The PVA-IA hydrogels were prepared with different elasticities ranging from 10.3 to 30.4 kPa storage moduli by controlling the crosslinking time with glutaraldehyde. Subsequently, rhVN was grafted on PVA-IA hydrogels with or without poly-L-lysine main chains for the adjustment of the surface charge of PVA-IA hydrogels. This study investigates the optimal elasticity of PVA-IA hydrogels grafted with rhVN that is prepared with much less concentration (5 g/ml) compared to the concentration of oligovitronectin (500 g/ml), which was used in previous study for the expansion of hPSCs for a long period of hPSCs culture (10-20 passages) under xeno-free condition. hPSCs cultured on PVA-IA hydrogels grafted with rhVN were evaluated from pluripotent protein expression by immunostaining, embryoid body (EB) formation, and teratoma formation after 10 and 20 passages. Keywords: pluripotent stem cell, Xeno-free, surface charge, hydrogel, recombinant vitronectin
