博碩士論文 105324019 詳細資訊




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姓名 潘智賢(Chih-Hsien Pan)  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 人類胚胎幹細胞在無滋養層及無異種條件的培養下於帶有生長因子的表面進行培養
(Human embryonic stem cells culture on hydrogels immobilized with human FGF-2)
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摘要(中) 人類多功能幹細胞再生醫跟再生醫學領域來說是一種非常有吸引力的的細胞,因為它們具有多能性以至於可以去分化成不同的人體細胞。所以如何去培養人類胚胎幹細胞已經成為一個熱門的主題。通常在多數的細胞研究中需要換培養液,且培養液中的生長因子需要儲存在非常低溫的冰箱下不然會降解跟變形。而降解跟變形的生長因子是造成培養人類多功能幹細胞的培養的成本很高的原因之一。 
在這個研究中把血管生成之纖維母細胞生長因子(FGF-2)嫁接在培養皿上有兩個目的。其一是讓細胞培養中減少培養液中FGF-2的用量;其二是讓生長因子嫁接在表面以至於可以使用比培養液懸浮的生長因子單位密度還要低還可以維持細胞的多能性。那為了確保實驗室成功的我們使用另外一種沒有FGF-2的培養液去培養細胞。目前使用兩種水凝膠來當作基底,嫁接了半胱氨酸與寡?。其中生長因子會與交聯劑鍵結後與半胱氨酸鍵結於水凝膠表面上。在目前的實驗中我們已經可以維持細胞的多能性且讓細胞可以培養超過代。我們堅信我們開發的材料表面應該是可以讓人類多功能幹細胞在培養中保持多能性。
短期目標是能讓人類多功能幹細胞能在生長因子嫁接的表面上維持多能性,最終目標是能夠嫁接不同生長因子促進人類多功能幹細胞分化成特定的細胞。
摘要(英) Human embryonic stem cells (hESCs) are an attractive prospect for regenerative medicine and tissue engineering because of their pluripotency to differentiate into different types of cells. Therefore, how to culture hPSCs has been to consider a very critical issues. Typically, the culture medium of hESCs contains growth factors such as FGF-2 and TGF-β1, which should be stored at -20 ~ -80 degree to prevent denaturation of the growth factors. This process is leading to the high cost of hESC culture medium.
In this experiment, we designed hESC culture dishes immobilized with FGF-2 to reduce the usage of FGF-2 in the culture medium. PVA-IA and CMC hydrogels were grafted with cysteine, which enables to graft the oligopeptide (VN2C) and growth factor (FGF-2) via crosslinker of PEG-SPDP. hESCs (H9) are expected to proliferate and maintain their pluripotency on modified hydrogel surface grafted with FGF-2 in the medium without using FGF-2. To verify this idea, hESCs were cultured on FGF-2 immobilized surface in Essential 6 cell culture medium plus TGF-β1, which corresponds to Essential 8 cell culture medium minus FGF-2. We added Essential 8 medium at first day to stabilize the hESCs to attach on the surface. hESCs culture on CMC-VN2C-FGF surface were found to keep their pluripotency after long term cultivation (>5 passages).
It should be a promising surface, which is immobilized growth factor on cell culture dishes for reducing usage of growth factor such as FGF-2 in the culture medium of hESCs as well as human induced pluripotent stem cells (hiPSCs). The next step is to immobilized the growth factors on the surface to control the hESCs differentiation into specific lineages of the cells.
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關鍵字(中) ★ 人類胚胎幹細胞
★ 培養
★ 水凝膠
★ 多能性
★ 生長因子
關鍵字(英) ★ human embryonic stem cell
★ culture
★ hydrogel
★ pluripotency
★ growth factor
論文目次 ABSTRACT
摘要 VI
INDEX OF CONTENT VII
INDEX OF FIGURE X
INDEX OF TABLE XV
Chapter 1 Introduction 1
1-1 Regenerative medicine 1
1-1-1 Stem cell therapy 2
1-2 Stem Cells 3
1-2-1 Differentiation potency of stem cell 4
1-3 Human pluripotent stem cells (hPSCs) 6
1-3-1 Human embryonic stem cells (hESCs) 6
1-3-2 Human induced pluripotent stem cells (hiPSCs) 7
1-3-3 Limitation of pluripotent stem cells in clinical trials 7
1-4 Cultivation of human pluripotent stem cells (hPSCs) 9
1-4-1 Extracellular matrix (ECM) coated dishes 10
1-4-2 Oligopeptide-grafted surface 13
1-5 Microenvironment effect for hPSCs culture 15
1-5-1 Elasticity of biomaterials for hPSCs cultivation 16
1-5-2 The effect of hydrophilicity of biomaterials on hPSCs adhesion and function 18
1-5-3 Cell-cell interaction and cell-ECM interaction 19
1-5-4 Soluble factors for hPSCs culture 20
1-6 Characteristic of hPSCs 27
1-6-1 First screening—Colony forming morphology 27
1-6-2 Second screening—Alkaline phosphatase (ALP) activity 28
1-6-3 Second screening—Expression of pluripotent proteins and genes 28
1-6-4 Third screening—Differentiation ability of hPSCs 28
1-7 Goal of this study 29
2-1 Materials 33
2-1-1 Cell line 33
2-1-2 Cell culture media, components, and buffer 33
2-1-3 Surface modification of hydrogels 35
2-1-4 Immunofluorescence staining of hESCs for pluripotency and differentiation assay 37
2-2 Method 39
2-2-1 Preparation of FGF-2 immobilized surface 39
2-2-2 hESCs culture and passage procedure 44
2-2-3 Characterization of FGF-2 immobilized surfaces 46
2-2-4 Characterization of cells growth, pluripotency expression, and differentiation ability in vitro and in vivo 47
Chapter 3 Results and Discussion 53
3-1 Screening of the optimal concentration of cysteine grafted on PVA-IA hydrogels immobilized FGF-2 53
3-1-1 Characterization of PVA-IA hydrogels immobilized FGF-2 with different concentration of cysteine by XPS analysis 54
3-1-2 Characterization of PVA-IA hydrogels immobilized FGF-2 with different concentration of cysteine by ELISA 57
3-1-3 hESCs cultured on PVA-IA hydrogels immobilized FGF-2 with different concentration of cysteine 59
3-2 PVA-IA hydrogels immobilized FGF-2 with increasing the FGF-2 concentration 64
3-2-1 Characterization of PVA-IA hydrogels immobilized FGF-2 with increasing FGF-2 concentration during conjugation reaction by XPS 65
3-2-2 ELISA of PVA-IA hydrogels immobilized FGF-2 with increasing FGF-2 concentration during conjugation reaction 67
3-2-3 hESCs cultivation on PVA-IA hydrogels immobilized FGF-2 with increasing FGF-2 concentration during conjugation reaction 70
3-3 o-Carboxylmethyl chitosan (CMC)-based hydrogels immobilized with FGF-2 75
3-3-1 Water contact angle measurements of CMC-based hydrogels grafted with FGF-2 76
3-3-2 XPS analysis of CMC-based hydrogels immobilized with FGF-2 78
3-3-3 FGF-2 evaluation of CMC-based hydrogels immobilized with FGF-2 by ELISA 84
3-3-4 hESCs cultivation on CMC-based hydrogels immobilized FGF-2 86
3-3-5 Pluripotent analysis of hESCs after long term cultivation on CMC-based hydrogels grafted with FGF-2 98
Chapter 4 Conclusion 106
References 108
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指導教授 劉青原(Ching-Yuan Liu) 審核日期 2018-8-13
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