人類多能幹細胞的培養與分化在 樹枝狀分子表面的熱響應水凝膠表面;Thermoresponsive Hydrogel Surface Grafted with Dendrimer Surface for Cultivation and Differentiation of Human Pluripotent Stem Cells

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Title:	人類多能幹細胞的培養與分化在樹枝狀分子表面的熱響應水凝膠表面;Thermoresponsive Hydrogel Surface Grafted with Dendrimer Surface for Cultivation and Differentiation of Human Pluripotent Stem Cells
Authors:	張家綸;Chang, Chia-Lun
Contributors:	化學工程與材料工程學系
Keywords:	N-異丙基丙烯酰胺;樹狀物;人類多能幹細胞;PNIPAAm;Dendrimer;hPSCs
Date:	2025-07-14
Issue Date:	2025-10-17 11:13:22 (UTC+8)
Publisher:	國立中央大學
Abstract:	人類多能幹細胞（hPSCs）在幹細胞治療中具有巨大潛力，並被用於心臟模型研究。近年來，已建立將 hPSCs 分化為心肌細胞的培養方法。然而，大多數細胞純化方法（如磁性活化細胞分選（MACS）、螢光活化細胞分選（FACS）和營養剝奪）在臨床應用中難以獲得足夠數量的高純度 hPSC 衍生心肌細胞。因此，為了簡化並改善這些複雜的細胞分選方法，我們開發了具有熱響應表面的細胞分選培養皿，其臨界溶解溫度約為 30°C。我們選擇了聚（N-異丙基丙烯醯胺）（PNIPAAm）作為熱響應高分子材料。在組織培養聚苯乙烯（TCP）培養皿上塗覆熱響應性 PNIPAAm 和細胞外基質（ECM）蛋白後，將 hPSCs 培養並分化為心肌細胞。接著，將培養皿冷卻至 4°C，保持 30 分鐘。隨後，在熱響應表面上收集（a）黏附力較弱而脫落的細胞和（b）黏附力較強而保留的細胞。為了製備熱響應培養皿，我們在 TCP 培養皿上製備了聚乙烯醇-衣康酸（PVA-IA）水凝膠。經過 N-(3-二甲基氨基丙基)-N’-乙基碳二亞胺鹽酸鹽（EDC）和 N-羥基琥珀酰亞胺（NHS）活化後，將聚（酰胺-胺）樹狀高分子（第 3 代，G3）共價結合到 PVA-IA 水凝膠上。接著，利用 PEG4-SPDP（琥珀酰亞胺 3-(2-吡啶二硫基) 丙酸酯）作為交聯劑，將羧基化 PNIPAAm（COOH-PNIPAAm-SH）共價接枝至 PVA-IA 水凝膠上的樹狀高分子氨基基團。之後，表面塗覆細胞外基質（ECM）蛋白（如重組玻連蛋白 rVN 或層粘連蛋白 LN-521），或進行活化以接枝多肽。當熱響應培養皿準備就緒後，我們將人誘導多能幹細胞（hiPSCs）接種於培養皿中，並在熱響應表面上誘導其分化為心肌細胞。隨後，將培養皿冷卻至4°C，保持 30 分鐘，使熱響應表面變為親水性。當表面親水性改變後，通過輕微搖晃或移液，脫離黏附力較弱的細胞，如心肌細胞。最後，利用流式細胞術分析心臟標誌蛋白 cTnT，評估熱響應表面對 hiPSC 衍生心肌細胞的分選效率。;Human pluripotent stem cells (hPSCs) are promising for stem cell therapy and are used in cardiac model studies. Recently, protocols for differentiating hPSCs into cardiomyocytes have been established. However, most of cell purification methods, such as Magnetic-activated cell sorting (MACS), Fluorescence-activated cell sorting (FACS), and nutrient depletion, are inefficient to purify a satisfactory number of high-purity hPSC-derived cardiomyocytes for clinical applications. To simplify and improve these complex cell sorting methods, we developed cell sorting dishes with thermoresponsive surfaces, which have a lower critical solution temperature of approximately 30°C. We selected poly(N-isopropyl acrylamide) (PNIPAAm), a thermoresponsive polymer. After culturing and differentiating hPSCs into cardiomyocytes on tissue culture polystyrene (TCP) plates coated with thermoresponsive PNIPAAm and extracellular matrix (ECM) protein, the plates were cooled to 4°C for 30 minutes. Subsequently, (a) detached cells with weak adhesion and (b) retained cells with strong adhesion were collected on the thermoresponsive surfaces. To prepare thermoresponsive dishes, polyvinylalcohol-itaconic acid (PVA-IA) hydrogels were prepared on tissue culture polystyrene (TCP) plates. After activation using N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysuccimimide (NHS), the poly(amidoamine) dendrimer (Generation 3, G3) was conjugated on PVA-IA hydrogels. (COOH)-PNIPAAm-(SH) was conjugated on amino group of dendrimer surface on PVA-IA hydrogels using crosslinker of PEG4-SPDP (succinimidyl 3-(2-pyridyldithio)propionate). Subsequently the surface was coated with extracellular matrix (ECM) proteins (recombinant vitronectin (rVN) or laminin-521 (LN-521)) or activated to graft peptides. Once the thermoresponsive dishes were ready, human induced pluripotent stem cells (hiPSCs) were seeded on the dishes. The hiPSCs were differentiated into cardiomyocytes on the thermoresponsive surface, and then, the surface of the dishes was cooled down to 4°C for 30 minutes, which generated the thermoresponsive dish surface became hydrophilic. Upon altering the surface hydrophilicity, the dishes were gently shook or pipetted to detach weakly adhered cells, such as cardiomyocytes. Lastly, the efficiency of cell sorting of hiPSC-derived cardiomyocytes on the thermoresponsive
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