熱敏性高分子塗佈基材表面設計連續培養系統應用於人類多能性幹細胞之增生;Design of Thermoresponsive Polymeric Surface for Continuous Culture of Human Pluripotent Stem Cells

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Title:	熱敏性高分子塗佈基材表面設計連續培養系統應用於人類多能性幹細胞之增生;Design of Thermoresponsive Polymeric Surface for Continuous Culture of Human Pluripotent Stem Cells
Authors:	林冠汝;Lin, Kuan-Ju
Contributors:	化學工程與材料工程學系
Keywords:	熱敏性;熱誘導細胞脫附;細胞培養;人類多能性幹細胞;Thermoresponsive;Thermally-induced cell detachment;Cell culture;Human pluripotent stem cells
Date:	2019-07-26
Issue Date:	2019-09-03 12:31:21 (UTC+8)
Publisher:	國立中央大學
Abstract:	人類多能性幹細胞 (human pluripotent stem cells (hPSCs))因具有自我增生能力以及分化能力，已成為現今再生醫學領域的新星。然而，臨床治療需要極大量的人類多性幹細胞，且典型的細胞培養是費時費力的批次培養。因此此研究設計連續培養系統以解決上述問題。人類胚胎幹細胞培養於熱敏性基材表面，並將溫度降低於熱敏性高分子的低臨界溶解溫度 (lower critical solution temperature (LCST)) 以改變基材表面性質以達到細胞部分脫附效果。未脫附細胞將繼續培養於原熱敏性基材上。此研究選用聚（N-異丙基丙烯酰胺 - 丙烯酸丁酯）(Poly(N-isopropylacrylamide-co-butylacrylate) (polyNIPAM-BA)) 為培養細胞之熱敏性高分子。研究不同濃度的 polyNIPAM-BA 塗佈於基盤對於溫度控制之地細胞貼附/脫附之影響，並找出最適當的塗佈濃度。並利用X射線光電子能譜儀 (XPS)、原子力顯微鏡 (AFM)以及表面對水接觸角量測等實驗鑑定以不同濃度之polyNIPAM-BA塗佈的基材表面。由細胞脫附實驗可知，最高的脫附率坐落在以8 mg/ml之polyNIPAM-BA塗佈的熱敏性基材。因此選用以8 mg/ml之polyNIPAM-BA塗佈之基材作為長期連續培養條件。本研究成功將人類多能性幹細胞，包括人類胚胎幹細胞(human embryonic stem cells (hESCs))以及人類誘導性多能性幹細胞(human induced pluripotent stem cells (hiPSCs))，培養於熱敏性基材(polyNIPAM-BA (8 mg/ml)-rVN (5 μg/ml))上，且人類多能性幹細胞可從熱敏性基盤上連續脫附5-7個循環，每個循環可達到60%的脫附率。此外，幹細胞於長期量續培養後保有原有的多功能性以及分化能力。此連續培養方式能簡化幹細胞培養所需之設備。且若此連續培養方式從2D培養方式到3D培養系統，將成為醫學應用於臨床治療領域的一大益處。 ;Human pluripotent stem cells (hPSCs), having the self-renewal ability and differentiation ability, have become promising candidates for regenerative medicine in these days. However, the number of the cells used for clinic treatment was large and the cell culture process was laborious. To solve these problems, the continuous culture system was developed in this study. hPSCs were cultured on the thermoresponsive surfaces and partially detached the cells by reducing the temperature below the lower critical solution temperature (LCST) of the thermoresponsive polymer. The remaining cells would be cultured in the same dishes by replacing the fresh medium. Poly(N-isopropylacrylamide-co-butylacrylate) (polyNIPAM-BA) was selected as thermoresponsive polymer for cell cultivation. The various concentration of polyNIPAM-BA was investigated for determining the optimal concentration of polyNIPAM-BA for thermally-modulated cell adhesion/detachment. The different substrate surfaces prepared by coating the various concentration of polyNIPAM-BA were analyzed the surface properties by water contact angle goniometer, X-ray photoelectron spectra (XPS) and atomic force microscope (AFM). According to the thermally-induced cell detachment process, the thermoresponsive surface coated with 8 mg/ml of polyNIPAM-BA showed the highest detachment ratio. Therefore, 8 mg/ml of polyNIPAM-BA was chosen for coated concentration of long-term cultivation substrate. In this study, hPSCs, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), were successfully cultured on the thermoresponsive substrates (polyNIPAM-BA (8 mg/ml)-rVN (5 μg/ml)) and partially detached from the thermoresponsive surfaces for 5-7 cycles. For each cycle, the partial detachment ratio could reach around 60 %. In addition, the cells indeed retained their pluripotency and differentiation ability after long-term cultivation. This continuous culture system might downsize the equipment requirement. Moreover, shifting two-dimensional culture to three-dimensional culture might be a great benefit for regenerative medicine in clinical treatment in the future.
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