連續式培養系統於接枝具奈米鏈段之熱敏感生物材料控制人類胚胎與誘導多功能幹細胞增生

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楊佳馨(Jia-Sin Yang) 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

連續式培養系統於接枝具奈米鏈段之熱敏感生物材料控制人類胚胎與誘導多功能幹細胞增生
(Continuous harvest system for expansion of human ES and iPS cells cultured on biomaterials immobilized with thermoresponsive nanosegments)

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摘要(中)

於再生醫學與組織工程之領域而言，人類多能性幹細胞具有吸引力的前景。典型培養細胞的方式大多為耗時與成本高的批次培養，本研究致力於發展連續收穫幹細胞之系統，利用低臨界溶解溫度(LCST)的熱敏感材料製備培養皿去培養幹細胞且降低培養液溫度使得細胞脫附，可維持細胞聚集和片狀，取代使用酵素讓細胞從培養皿上脫附。
熱敏感奈米片段表面由三種共聚物製備而成，利用可逆加成斷裂鏈轉移(RAFT)高分子合成法製備三種具有聚苯乙烯(polystyrene)的共聚物為(a)熱敏感性的聚(N-異丙基丙烯?胺)(poly[styrene-co-N-isopropylacrylamide], PNIPAAm)(b)生物相容性的聚乙二醇甲基丙烯酸酯(poly[styrene-co-polyethylene glycol methacrylate], PEGMA)和(c)可藉由聚丙烯酸(polyacrylic acid, PAA)的羧酸基(carboxylic acid)共軛寡?(oligo-vitronectin)。
本研究成功將人類胚胎幹細胞與人類誘導多能性幹細胞培養於熱敏感表面且可連續培養7個循環，藉由螢光免疫染色、胚體體外分化及畸形瘤的體內生成分析，得知經由培養7個循環後，人類胚胎幹細胞與人類誘導多能性幹細胞依然維持其多能性及分化能力。此外，將培養皿置於銅板上且放進7-9度的冰箱中，30分鐘之內hESCs與hiPSCs脫附率可超過60%，此連續培養方式能縮小幹細胞培養所需的設備且簡化培養過程，同時也可避免使用酵素脫附對細胞造成的傷害，細胞可以維持其多能性。還可通過將2D培養方式轉移到3D新型培養以獲得更大量的細胞數量，這對於再生醫學的臨床應用上將會是非常有益的。

摘要(英)

Human pluripotent stem cells (hPSCs) are an attractive prospect for regenerative medicine and tissue engineering. Typical stem cell cultivation methods are still based on batch-type culture, which is laborious and expensive. In this study, It is developed that the partial detachment stem cell culture system by using thermoresponsive nanosegments, a polymer having low critical solution temperature (LCST), which were coated on the surface of cell culture dishes for continuous stem cell harvest. This method enables cell aggregates or cell sheets to be obtained in culture medium without applying an enzymatic digestion.
The themoresponsive nanobrush surfaces are composed by three copolymers having polystyrene, which are (a) thermoresponsive poly(N-isopropyl acrylamide), PNIPAAm, (b) biocompatible polyethylene glycol methacrylate (PEGMA) and (c) polyacrylic acid (PAA) where bioactive oligopeptide (oligo-vitronectin) could be conjugated via carboxylic acid of PAA. P[St-NIPAAm] (poly[styrene-co-N-isopropylacrylamide]), and P[St-PEGMA] (poly[styrene-co-polyethylene glycol methacrylate]) were prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization.
In this study, I successfully cultured human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) on the thermoresponsive surface and continuously harvested for seven and ten cycles respectively. The hESCs and hiPSCs have high pluripotency and differentiation ability by immunostaining analysis, embryoid body (EB) formation and teratoma formation after seven cycles. In addition, over 60% of hESCs and hiPSCs could be detached from the surface after 30 minutes of low temperature incubation (7-9°C). This continuous culture system prevents hESCs and hiPSCs from enzymatic digestion damages and allows cells maintain their pluripotency on the surface. The continuous harvest of stem cells should downsize the equipment requirements for stem cell culture and simplify the culture process. Moreover, This cultivation method also can be scaled up cell numbers by shifting 2D to a novel 3D culture system, which will be a great benefit to its clinical application in regenerative medicine.

關鍵字(中)

★ 幹細胞
★ 熱敏感
★ 奈米片段
★ 細胞培養
★ 胚胎幹細胞

關鍵字(英)

★ Stem cell
★ Thermoresponsive
★ Nanosegment
★ Cell culture
★ Embryonic stem cell

論文目次

Abstract I
摘要 III
Index of Figure VI
Index of Table XVI
Chapter 1. Introduction 1
1-1 Stem Cell Applications in Regenerative Medicine 1
1-2 Stem Cells 3
1-2-1 Pluripotent Stem Cells (PSCs) 6
1-3 Human Pluripotent Stem Cells (hPSCs) Cultivation (2D) 9
1-3-1 hPSC Culture Under Xeno-Free Conditions 10
1-3-2 hPSCs Culture on Thermoresponsive Surface 15
1-4 Three-dimensional (3D) Cultivation of hPSCs 27
1-4-2 hPSCs Culture on Thermoresponsive Materials for 3D Culture 33
1-5 Characterization of human Pluripotent Stem Cells (hPSCs) 36
1-5-1 Colony Formation 37
1-5-3 Alkaline phosphatase activity 38
1-5-2 Pluripotent Gene Expression 38
1-5-3 Pluripotent Protein Expression 38
1-5-4 Differentiation Ability 39
Chapter 2. Materials and Methods 42
2-1 Materials 42
2-1-1 Cell Lines 42
2-1-2 Commercial Coating Dishes 42
2-1-3 Materials for Thermoresponsive Surface 42
2-1-4 Medium and Others 43
2-1-5 Immunostaining Materials 44
2-2 Methods 45
2-2-1 Preparation of Thermoresponsive Dishes (2D) 45
2-2-3 hPSCs Culture on the Thermoresponsive Dishes (2D) 49
2-2-4 hPSCs Adhesion and Detachment 50
2-2-5 Differentiation Ratio of hPSCs Colonies 51
2-2-6 Continuous Culture System of hPSCs 52
2-2-7 Characterization of Thermoresponsive Surface 53
2-2-8 Characterization of hPSCs 54
2-2-9 Three-dimensional (3D) Culture of hESCs 59
Chapter 3. Results and Discussion 62
3-1 Characterization of Thermoresponsive Surface 62
3-1-1 X-ray photoelectron spectroscopy (XPS) analysis 63
3-1-2 Water Contact Angle Analysis of the PNIPAAm Coating Surface 67
3-2 Continuous Culture of hPSCs on the Thermoresponsive Surface 70
3-2-1 hESCs Cultivated on the Thermoresponsive Surface Treated with Roller Cutter 70
3-2-2 hESCs Cultivated on Thermoresponsive Surface with Pipetting 88
3-2-3 Continuous Culture of hiPSCs on Thermoresponsive Surface by re-coating rVN every two cycles 106
3-3 Characterization of hPSCs after Long-term Cultivation 113
3-3-1 Immunostaining Analysis 113
3-3-2 Differentiation Ability in Vitro 116
3-3-3 Differentiation Ability in Vivo 119
3-3-4 Cardiomyocyte Differentiation of hESCs after Long-term Culture 120
3-4 hESCs Cultivated on the Thermoresponsive Microcarrier Beads 126
3-4-1 X-ray photoelectron spectroscopy (XPS) analysis 126
3-4-2 Continuous Culture of hESCs on the Thermoresponsive Microcarrier beads 130
Chapter 4. Conclusion 136
Reference 138
Supplementary Data 154

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指導教授

?口亞紺(Akon Higuchi)

審核日期

2018-7-27

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