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

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姓名

林冠汝(Kuan-Ju Lin) 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

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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★ 羊水間葉幹細胞培養於接枝細胞外間質寡肽與環狀肽具有最佳表面硬度的生醫材料,其增殖能力及多能性之研究	★ 人類體細胞從組成誘導型多能性幹細胞培養在無飼養層上

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至系統瀏覽論文 (2024-7-26以後開放)

摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ 熱敏性
★ 熱誘導細胞脫附
★ 細胞培養
★ 人類多能性幹細胞

關鍵字(英)

★ Thermoresponsive
★ Thermally-induced cell detachment
★ Cell culture
★ Human pluripotent stem cells

論文目次

摘要 I
Abstract II
致謝 IV
Index of Content V
Index of Figure VIII
Index of Table XV
Chapter 1. Introduction 1
1-1 Stem cells 1
1-1-1 Pluripotent stem cells (PSC) 5
1-1-1-1 Embryonic stem cells (ESCs) 5
1-1-1-2 Induced pluripotent stem cells (iPSCs) 7
1-2 Microenvironment effect on stem cells 9
1-2-1 Extracellular matrix (ECM) 10
1-2-2 Soluble factors 11
1-2-3 Cell-cell interactions 13
1-2-4 Physical surfaces parameters 14
1-2-5 Effect of physiochemical environment on hPSC culture 18
1-3 Conventional cultivation of hPSCs 18
1-3-1 Chemically defined culture medium 19
1-3-2 Chemically defined biomaterials for 2D cultivation 21
1-3-2-1 hPSCs cultured on ECM-immobilized surfaces 23
1-3-2-2 hPSCs culture on oligopeptide-immobilized surfaces 24
1-3-2-3 hPSCS culture on synthetic materials 25
1-3-3 Three dimensional hPSCs cultivation 27
1-4 Smart, thermally-induced biomaterials for bio-application 31
1-4-1 The preparation of thermoresponsive surfaces 32
1-4-2 The properties of thermoresponsive surfaces 35
1-4-3 The application of PIPAAm and its derivatives-modified substrates 39
1-4-3-1 Cells sheet formation 39
1-4-3-2 Cells and biomolecules separation 41
1-4-3-3 3D cell culture system by thermoresponsive microcarriers 43
1-5 Characterization of pluripotent stem cells 45
1-5-1 Colony formation 46
1-5-2 Alkali phosphatase activity 46
1-5-3 Pluripotency evaluation 46
1-5-4 Differentiation ability evaluation 47
1-6 Goal of the study 49
Chapter 2. Materials and Methods 50
2-1 Materials 50
2-1-1 Cell lines 50
2-1-2 Cell culture media, buffer and others 50
2-1-3 Cell culture substrates 51
2-1-4 Materials for hPSCs characterization (Immunostaining and teratoma assay) 52
2-2 Experimental instruments 53
2-3 Methods 54
2-3-1 hPSCs culture and passage procedure 54
2-3-1-1 Cell culture and passage 54
2-3-1-2 Cell cryopreservation 55
2-3-1-3 Cell thawing 55
2-3-2 Preparation procedure of thermoresponsive substrates 56
2-3-2-1 Preparation of two-dimensional thermoresponsive dishes 56
2-3-2-2 Preparation of three-dimensional thermoresponsive microcarriers 57
2-3-3 Substrate surfaces characterization 58
2-3-3-1 Contact angle goniometer (CA) 58
2-3-3-2 X-ray photoelectron spectra (XPS) 58
2-3-3-3 Atomic force microscope (AFM) 59
2-3-4 Continuous cultivation of hPSCs on thermoresponsive substrates 59
2-3-4-1 Continuous cultivation of hPSCs on 2D thermoresponsive dishes 59
2-3-4-2 Continuous cultivation of hPSCs on 3D thermoresponsive microcarriers 62
2-3-5 Characterization of cells growth, pluripotency expression, and differentiation ability in vitro and in vivo 63
2-3-5-1 Cell density measurement 63
2-3-5-2 Expansion fold and doubling time of hPSCs 64
2-3-5-3 Differentiation ratio 65
2-3-5-4 Immunofluorescence staining 65
2-3-5-5 Differentiation ability in vitro assay: Embryo body (EB) formation 68
2-3-5-6 Differentiation ability in vivo assay: teratoma formation 69
Chapter 3. Results and Discussion 70
3-1 Characterization of the thermoresponsive surface 70
3-1-1 X-ray photoelectron spectroscopy (XPS) analysis 70
3-1-2 Water contact angle (CA) analysis 73
3-1-3 Atomic force microscope (AFM) 77
3-2 Continuous culture of hPSCs on the thermoresponsive surfaces 79
3-2-1 The optimal concentration of polyNIPAM-BA for thermal detachment of hPSCs 80
3-2-2 Continuous cultivation of hPSCs on the thermoresponsive surfaces 86
3-2-2-1 hESCs cultured on the thermoresponsive surfaces continuously 87
3-2-2-2 hiPSCs continuously cultured on the thermoresponsive surfaces 91
3-3 Characterization of hPSCs after long-term cultivation on thermoresponsive surface 95
3-3-1 Immunostaining analysis of pluripotency on hPSCs 95
3-3-2 Immunostaining analysis of differentiation ability in vitro 97
3-4 hPSCs cultured on Thermoresponsive microcarriers 99
3-4-1 X-ray photoelectron spectroscopy (XPS) analysis 99
3-4-2 Continuous cultivation of hESCs on thermoresponsive microcarriers 102
Chapter 4. Conclusion 106
References 108
Supplementary Data 120

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指導教授

樋口亞紺

審核日期

2019-7-26

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