博碩士論文 104324049 詳細資訊




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姓名 陳麗華(Li-Hua Chen)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 藉由調整水凝膠之表面電荷及軟硬度並嫁接玻連蛋白用以培養人類多功能幹細胞
(Human Pluripotent Stem Cells Cultured on Recombinant Human Vitronectin-Grafted Hydrogels by Adjusting Surface Charge and Elasticity)
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摘要(中) 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
摘要(英)
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
關鍵字(中) ★ 多能性幹細胞
★ 無異種
★ 表面電荷
★ 水凝膠
★ 玻連蛋白
★ 軟硬度
關鍵字(英) ★ pluripotent stem cell
★ xeno-free
★ surface charge
★ hydrogel
★ recombinant vitronectin
★ elasticity
論文目次
Index of Content
Abstract I
摘要 III
Index of Content IV
Index of Figure VII
Index of Table XII
Chapter 1 Introduction 1
1-1 Stem Cells 1
1-1-1 Human Embryonic Stem Cells (hESCs) 2
1-1-2 Human Induced Pluripotent Stem Cells (hiPSCs) 4
1-2 Expansion of Pluripotent Stem Cell under Xeno-Free Conditions 5
1-2-1 hPSCs Culture on ECM-Coated Surface (2D) 6
1-2-2 hPSCs Culture on Oligopeptide-Immobolized Surface 8
1-2-3 hPSCs Culture on Synthetic Polymer Surface (2D) 10
1-3 Microenvironment Effect on hPSCs 13
1-3-1 The Effect of Biomaterial Elasticity on hPSCs 14
1-3-2 The Effect of Hydrophilicity of Biomaterials on hPSCs Adhesion and Function 21
1-3-3 The Effect of Biomaterial Surface Charges on hPSCs 21
1-3-4 Cell-Cell Interactions 22
1-4 Characterization of hPSCs 22
1-4-1 Colony Formation 24
1-4-2 Alkali Phosphatase Activity 24
1-4-3 Pluripotent Gene Expression 24
1-4-4 Pluripotent Protein Expression 24
1-4-5 Differentiation Ability 25
1-4-6 Immunofluorescence 27
1-5 Cardiomyocyte Differentiation 28
1-5-1 Effects of Elasticity of Biomaterials on The Differentiation of hPSCs into Cardiomyocytes 29
1-5-2 Effects of Biomaterial Surface on The Cardiomyocytes Adhesion 30
1-5-3 Characterization of Cardiomyocytes 31
Chapter2. Material and Methods 36
2-1 Material 36
2-1-1 Cell Line 36
2-1-2 Commercial Coated Dishes 36
2-1-3 Medium and Others 36
2-1-4 Chemical Materials 37
2-1-5 Immunostaining 41
2-2 Cell Culture 42
2-2-1 Preparation of Cross-Linked PVA-IA Hydrogel Dishes 42
2-2-2 Preparation of PVA-IA Hydrogel Dishes Grafted with PLL and rVN 43
2-2-3 hESCs Culturing and Passaging 44
2-2-4 Cryopreservation of hESCs 45
2-2-5 hESCs Thawing 45
2-2-6 Cardiomyocyte Differentiation under Xeno-Free Condition 46
2-3 Characterization of Dish Surface 47
2-3-1 X-ray Photoelectron Spectra 47
2-3-2 Contact Angle Goniometer 47
2-3-3 Zeta Potential Measurements of Hydrogel Surface 48
2-3-4 Zeta-Potential Measurement of Polymer Solution 48
2-4 Characterization of hESCs 49
2-4-1 Cell Density Measurements 49
2-4-2 Expansion Fold and Doubling Time of hPSCs 50
2-4-3 Differentiation Ratio of hPSCs 50
2-4-4 Immunostaining of Cells 51
2-4-5 Embryoid Body Formation 52
2-4-6 Teratoma Formation 54
2-5 Characterization of hESCs-Derived Cardiomyocytes 54
2-5-1 Sarcomere Immunostaining Analysis 54
2-5-2 Flow Cytometry Analysis 56
Chapter 3. Results and Discussion 57
3-1 PVA-IA Films Grafted with 10μg/ml of rVN with Different Elasticities 57
3-1-1 Physical Characterization of PVA-IA Films 57
3-1-2 Characterization of PVA-IA Films Grafted with 10μg/ml of rVN with Different Elasticities 59
3-1-3 Cultivation of hESCs on PVA-IA Films Grafted with 10μg/ml of rVN with Different Elasticities 63
3-2 PVA-IA-24H Films Grafted with Different Concentration of rVN 69
3-2-1 Characterization of PVA-IA-24H Films Grafted with Different Concentration of rVN 69
3-2-2 Cultivation of hESCs on PVA-IA-24H Films Grafted with Different Concentration of rVN 73
3-2-3 The Effect of Hydrophilicity of Biomaterials on hESCs Adhesion and Function 76
3-2-4 The Effect of Biomaterial Surface Charges on hPSCs Culture 80
3-3 PVA-IA Films Grafted with Poly-L-Lysine and 5μg/ml of rVN 85
3-3-1 Characterization of PVA-IA Films Grafted with Poly-L-Lysine and 5μg/ml of rVN 85
3-3-2 Cultivation of hESCs on PVA-IA Films Grafted with PLL and 5μg/ml of rVN with Different Elasticities 87
3-4 Pluripotency Analysis of hESCs After Cultivation on PVA-IA Films Grafted with PLL and rVN Having Different Elasticities for 10 passages. 91
3-4-1 Immunostaining of Pluripotent proteins 91
3-4-2 Differentiation Ability In Vitro – Embryoid Body Formation 98
3-5 The Effect of Elasticity of Biomaterials on The Differentiation of hESCs into Cardiomyocytes 105
Chapter 4. Conclusions 111
Reference 113
Supplementary Data 121
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指導教授 樋口亞紺(Akon Higuchi) 審核日期 2017-8-22
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