用於人類胚胎幹細胞生長之生醫材料其奈米片段分子之設計

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曾也家(Yeh-Chia Tseng) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

用於人類胚胎幹細胞生長之生醫材料其奈米片段分子之設計
(Molecular Design of Nanosegments on Cell Culture Biomaterials for Proliferation of Human Embryonic Stem Cells)

相關論文

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至系統瀏覽論文 (2028-7-27以後開放)

摘要(中)

幹細胞於再生醫學以及組織工程之應用上是相當具有前瞻性的研究來源。其中，人類胚胎幹細胞以其能保有細胞原有之多功能特性而常用於生醫領域上之研究。常見的塗層培養材料，如：基質膠、層黏蛋白511、層黏蛋白521、重組玻連蛋白等等。這些塗層材料皆是以細胞分泌之細胞外間質而來。在本研究中，有三組不同的奈米片段設計，用以探索人類胚胎幹細胞於無異種材料上之生長，包含：(一)以聚（乙二醇）2-氨基乙基醚乙酸交聯劑接枝於聚（乙烯醇 - 共 - 衣康酸）水凝膠與寡玻?之間之材料 (二)於聚（乙烯醇 - 共 - 衣康酸）水凝膠接枝含不同長度之結合片段之寡?之材料 (三)可塗層於培養盤之奈米片段設計之材料，以上材料將透過鑑定分析評估其在生物醫學領域上之應用。
(一)以聚（乙二醇）2-氨基乙基醚乙酸交聯劑接枝於聚（乙烯醇 - 共 - 衣康酸）水凝膠與寡玻?間，為了使先前的研究能得以改善其表面所需寡?濃度高之問題(高於500μg/ml)，本研究中利用交聯劑特性於表面提供了一長鏈結構，一則使得表面結構更加地具有彈性，便於細胞貼附時更容易找到寡?上之鍵結部位，使得表面所需之寡?含量降低。二則能模仿如同細胞外間質蛋白上之微環境供細胞更好地貼附及生長。
(二)於聚（乙烯醇 - 共 - 衣康酸）水凝膠接枝含不同長度之結合片段之寡?之設計，包含：單鏈寡?、含有結合片段之單鏈寡?、雙鏈寡?以及含有結合片段之雙鏈寡?，透過設計以上序列以探索不同結合片段於寡?上所造成之影響，並藉由人類胚胎幹細胞培養於此材料上做多能性測試以及誘導成為心肌細胞以評估其在無異種環境下生醫領域之應用特性。
(三)為了改善小分子寡?接枝於聚（乙烯醇 - 共 - 衣康酸）水凝膠時所需之高濃度用量(高於500μg/ml)，希冀將疏水特性之寡?鏈連接於寡玻?之上以此模仿細胞外間質能塗層於培養盤之表面的特性，提供一種更為方便製作培養盤之方法。
以上奈米片段分子之設計，提供了三種於使用小分子寡玻?製作培養盤時之改良方法，以能使得小分子寡?能更好地運用於幹細胞培養以及再生醫學之臨床應用，並透過X射線光電子能譜、細胞型態之觀察以及免疫蛋白染色等，完整的評估其特性。

摘要(英)

Stem cells are an attractive prospect for regenerative medicine and tissue engineering. The dishes coated with Matrigel, Synthemax II, Laminin 511, Laminin 521, and Cellstart are typically used for the cell culture substrates for human embryonic stem cells (hESCs). These coating materials were based on extracellular matrix (ECM), which were secreted from cells. Our previous study showed that hESCs can be cultured on polyvinylalcohol-co-itaconic acid (PVA-IA) hydrogels conjugated with higher concentration of oligovitronectin (>500 μg/mL) as hESC culture substrates. In order to solve the reason of the higher concentration usage of oligovitronectin conjugated on PVA-IA hydrogels, we designed another PVA-IA hydrogels conjugated with oligovitronectin for hESCs culture substrates using crosslinker of PEG-AEAC (Poly(ethylene glycol)-2-aminoethyl ether acetic acid) and we evaluated hESC attachment and pluripotency cultured on these hydrogels. The PVA-IA hydrogels were prepared by coating aqueous PVA-IA solution on the tissue culture polystyrene (TCPS) dishes and were crosslinked with glutaraldehyde to control hydrogel stiffness. Twice activation was performed to conjugate oligovitronectin on PVA-IA hydrogels via PEG-AEAC crosslinker by the peptide bonding reaction between carboxylic group and amino group. We investigated the effect of long crosslinking agent of PEG-AEAC on the culture of hESCs on PVA-IA hydrogels grafted with oligovitronectin via PEG-AEAC. We have also tried same reaction but with different length of PEG-AEAC to evaluate the length effect on the culture of hESCs in the dishes. After the long term culture of hESCs on the PVA-IA hydrogels (more than 10 passages), we evaluated pluripotency and differentiation ability of hESCs. Moreover, we design several oligopeptide sequence with different length of joint segment to investigate which oligopeptide-grafted hydrogel molecular design with optimal length of joint segments supported the proliferation of hESCs while hESCs maintained their pluripotency and differentiation ability into cardiomyocytes under xeno-free cell culture conditions. In addition, we designed the small molecules of oligopeptides for hESC culture, which could be coated directly to the TCPS dishes in order to decrease the concentration of these small molecules of oligopeptides used and also to provide a much more convenient way to prepare hESC culture dishes.

關鍵字(中)

★ 人類胚胎幹細胞
★ 奈米片段
★ 細胞培養
★ 無異種
★ 分子設計
★ 生醫材料

關鍵字(英)

★ human embryonic stem cells
★ molecular design
★ nanosegments
★ xeno-free
★ cell culture
★ biomaterials

論文目次

Index of Content
Abstract I
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) 1
1.1.2 Human Induced Pluripotent Stem Cells (hiPSCs) 3
1.2 Expansion of Human Pluripotent Stem Cells under Xeno-Free Conditions 5
1.2.1 hPSCs Cultured on Surface Coated with ECM and ECM-mimicking Peptides in 2-D Culture 6
1.2.2 hPSCs Cultured on Oligopeptides-Immobilized Surface in 2-D Culture 11
1.2.3 hPSCs Cultured on Synthetic Polymer Surface in 2-D 15
1.3 Microenvironment Effects on hPSCs 16
1.3.1 The Effects of Biomaterials Elasticity for Differentiation Fates of hPSCs 17
1.3.2 The Effects of Biomaterial Hydrophilicity on hPSC Adhesion and Function 19
1.3.3 Cell-Cell Interaction 20
1.4 Characterization of hPSCs 22
1.4.1 Colony formation 23
1.4.2 Alkali Phosphatase Activity (ALP) 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 Differentiation of hPSCs into Cardiomyocytes 28
1.5.1 Efficient Methods for Differentiating hPSCs into Cardiomyocytes 29
1.5.2 Effect of Cell Culture Biomaterials on hPSC Differentiation into Cardiomyocytes 32
1.6 The goal of this study 33
Chapter 2 Materials and Method 35
2.1 Materials 35
2.1.1 Cell Line 35
2.1.2 Commercial Culture Dishes 35
2.1.3 Commercial Coated Substrates 35
2.1.4 Medium and Others 35
2.1.5 Chemical Materials 36
2.1.6 Immunostaining 47
2.2 Cell culture 48
2.2.1 Preparation for PVA Hydrogel Dishes Conjugated with PEG Crosslinker and Oligo-peptides 48
2.2.2 Preparation for PVA-IA Hydrogels Grafted with Oligo-peptides Having Different Length of Joint Segment 50
2.2.3 Preparation for Oligo-peptides Nanosegments Coating Dishes 52
2.2.4 Culture Method of hESCs 52
2.2.5 Passage Method of hESCs 53
2.2.6 Cryopreservation of hESCs 53
2.2.7 hESCs Thawing 54
2.3 Characterization of Dish Surface 55
2.3.1 Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy 55
2.3.2 X-ray Photoelectron Spectroscopy (XPS) 55
2.3.3 Dynamic Light Scattering (DLS) 56
2.4 Characterization of hESCs 56
2.4.1 Expansion Fold and Doubling Time of hPSCs 56
2.4.2 Differentiation Ratio of hPSCs 57
2.4.3 Immunostaining of Cells 57
2.4.4 Embryoid Body Formation 59
2.4.5 Teratoma Formation 60
2.5 Cardiomyocyte Differentiation 61
2.5.1 Cardiomyocyte Differentiation Method 61
2.5.2 Flow Cytometry Analysis 62
Chapter 3 Results and Discussion 63
3.1 PVA-IA Hydrogel Dishes Conjugated with PEG Crosslinker and Oligopeptides 63
3.1.1 Characterization of PVA-IA Hydrogels Conjugated with PEG Crosslinker and Oligopeptides 63
3.1.2 Cultivation of hESCs on PVA-IA Hydrogels Conjugated with PEG Crosslinker and Oligo-peptides 71
3.2 PVA-IA Hydrogels Grafted with Oligo-peptides Having Different Length of Joint Segment 78
3.2.1 Characterization of PVA-IA Hydrogels Conjugated with PEG Crosslinker and Oligopeptides 78
3.2.2 Cultivation of hESCs on PVA-IA Hydrogels Grafted with Oligo-peptides Having Different Length of Joint Segment 84
3.3 Oligopeptides Nanosegments Coating Dishes 107
3.3.1 Characterization of Oligopeptides Nanosegments Coating Dishes 107
3.3.2 Cultivation of hESCs on Oligopeptides Nanosegments Coating Dishes 122
Chapter 4 Conclusion 128
Supplementary Data 131
Reference 141

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指導教授

?口亞紺(Akon Higuchi)

審核日期

2018-7-30

推文