人類幹細胞培養於熱敏感奈米片段材料之研究

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姓名

彭依加查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

人類幹細胞培養於熱敏感奈米片段材料之研究
(Human stem cell culture on biomaterials immobilized with thermoresponsive nanobrush)

相關論文

★ 抗癌藥物的鑑定性用於分析大腸癌中癌幹細胞之研究	★ 利用具有奈米片段的生醫材料進行純化及去除癌症幹細胞
★ 羊水間葉幹細胞培養於細胞外間質及材料硬度/彈性表面,其分化能力及多能性之研究	★ 人類脂肪幹細胞的膜純化法與分化能力研究
★ 人類羊水間葉幹細胞培養於具有奈米片段與最佳表面硬度的生醫材料，其增殖與成骨分化能力	★ 多能幹細胞在無異種條件下分化為間充質幹細胞的生物材料比較研究

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摘要(中)

熱敏感材料表面因其熱敏感材料之低臨界溶液溫度 (LCST) 之特性使其成為細胞培養目前最引人矚目的一項技術。在細胞繼代時，我們可排除酵素的繼代方法而減少對細胞的傷害，改由降低溫度至4℃，使細胞成層(cell sheet)從表面脫附。這個設計主要有四個優點：一、建立一個非異種的培養系統，在人類幹細胞臨床使用上，可排除免疫排斥的疑慮；二、使用非酵素傷害的機制，改由降低溫度來達到細胞的脫附效果；三、此培養盤是利用高分子塗佈製成，在溫度改變之後並不會破壞其表面的構造，故可以重複使用，降低成本；四、以高分子塗佈的培養表面可做成微載體(Microcarrier)，培養幹細胞於生物反應器(Bioreactor)中，使其大量培養以應用至醫學組織與器官之培養與移植。此系統主要設計對象是針對人類幹細胞，包含人類胚胎幹細胞(hESCs)以及人類脂肪幹細胞(hADSCs)。我們使用的高分子尾端都接上一個聚苯乙烯端(polystyrene anchor)，聚苯乙烯端可穩定的和培養盤(TCPS)結合，而另一端則分別接上：(1) 熱敏感材料poly(N-isopropyl acrylamide), PNIPAAm； (2) 具有生物相容性質的polyethylene glycol methacrylate (PEGMA) ；以及 (3) polyacrylic acid (PAA) 可利用其上之羧基與寡肽鏈結合 (oligo-vitronectin)。這些塗佈的共聚物具有很小的分子量分布(小於1.6)，而後我們利用NMR以及FTIR來檢測其表面特性，共聚物塗佈的表面密度由XPS 以及 XPR來檢測。
我們將人類脂肪幹細胞培養在此共聚物塗佈的熱敏感奈米片段表面上以獲得其最適條件，其最適條件包含高貼附率以及高脫附效率。在低溫處理的過程中，最適化條件可達80% 之脫附效率，而控制條件TCPS盤上只有少於20%的細胞脫附。我們進而將此最適化條件培養人類胚胎幹細胞，人類胚胎幹細胞不只被成功培養於熱敏感奈米片段表面且他們可以維持其多能性。往後，我們希望能夠將此系統轉移至3D培養，利用先前所述之微載體，使其能培養及放大細胞量，在生物醫學應用方面有所貢獻。

摘要(英)

Thermoresponsive surface prepared using thermoresponsive polymers with low critical solution temperatures (LCSTs) is attractive candidates for cell culturing because cells can be detached from the surface without applying an enzymatic digestion method and, instead, by decreasing the temperature, e.g., to 4 °C, which enables cell aggregates or cell sheets to be obtained. In this study, the thermoresponsive nanobrush surfaces are designed for human stem cell culture (human adipose-derived stem cells [hADSCs] and human embryonic stem cells [hESCs]). Using RAFT polymerization, I prepared the coating copolymers having polystyrene anchor and (a) thermoresponsive poly(N-isopropyl acrylamide), PNIPAAm, (b) biocompatible polyethylene glycol methacrylate (PEGMA), and (c) polyacrylic acid (PAA) where bioactive oligopeptide (oligo-vitronectin) can be conjugated via carboxylic acid of PAA. The coating copolymers had narrow molecular weight distribution (PDI is less than 1.8) and were characterized by NMR and FTIR. The coating surface density was analyzed by XPS and SPR measurements. hADSCs were cultured on the surface coated with copolymers containing PNIPAAm, PEGMA and PAA conjugated with oligo-vitronectin. I investigated the optimization of hADSCs attachment and detachment with high efficiency in cooling process. While less than 20% of hESCs were able to detach from the commercially available control dishes, TCPS, hESCs can be cultured and detached from thermoresponsive nanobrush surfaces and maintained the pluripotency. Furthermore, hPSCs culture on thermorespsonsive nanobrush surfaces can be applied to 3D culture system for clinical application by introducing the microcarriers, which can be coated with copolymers developed in this study.

關鍵字(中)

★ 熱敏感材料
★ 人類幹細胞

關鍵字(英)

★ stem cell
★ thermoresponsive

論文目次

Chapter 1. Introduction 1
1-1 Cell therapy for regenerative medicine 1
1-1-1 Temperature-responsive cell culture surface……………………………… 2
1-1-2 Rapid recovery of cell sheets and cell sheet manipulation technology 5
1-1-3 Functionalization of temperature-responsive cell culture surfaces 8
1-1-4 Micropatterned temperature-responsive polymer brush surfaces for fabricating cell sheets with well-controlled orientational structures by RAFT 11
1-2 Stem cells 12
1-2-1 Self renewal and lineage plasticity of stem cells 14
1-2-2 Embryonic stem cells (ESCs) 16
1-2-3 Induced pluripotent stem cells 16
1-2-4 Adult stem cells 18
1-3 Microenvironment effect on human pluripotent stem cells 20
1-3-1 Environmental factors 21
1-3-2 Chemically defined materials for stem cell culture 23
1-4 Three-dimensional (3D) culture strategies for hPSCs bioprocesses 34
1-4-1 3D Culture of hPSCs on microcarriers 36
1-4-2 3D Culture of hPSCs entrapped in hydrogels (Microcapsules) 37
1-5 Characterization of pluripotent stem cells 41
1-5-1 Colony formation 41
1-5-2 Alkali phosphatase activity 41
1-5-3 Pluripotent gene expression 43
1-5-4 Pluripotent protein expression 43
1-5-5 Differentiation ability 43
1-5 Immunofluorescence 46
Chapter 2. Materials and Methods 48
2-1 Materials 48
2-1-1 Cells and culture media 48
2-1-2 Chemicals 48
2-2 Methods 52
2-2-1 Cell isolation of hADSCs 52
2-2-2 Preparation of the thermoresponsive nanobrush surfaces. 54
2-2-3 Culture medium preparation 55
2-2-4 10x Phosphate buffer saline (PBS) preparation 56
2-2-5 Cell culture 56
2-2-6 XPS analysis of nanobrush surfaces 58
2-2-7 Cell adhesion and detachment 59
2-2-8 Colony differentiation ratio 60
2-2-9 Cell proliferation assay 60
2-2-10 Immunofluorescence 62
Chapter 3 Results and Discussion 66
3-1 Characterization of thermoresponsive nanobrush surfaces 66
3-2-1 Effect of attachemnt/detachment of hADSCs on the surface having different chain length of PS-b-PAA and PS-b-PNIPAAM 74
3-2-2 Effect on varing ratio of PS-b-PNIPAAM and PS-b-PEGMA in hADSCs 78
3-2-3 Effect of surface coverage of PS-b-PAA on attachment/detachment of hADSCs 84
3-3 hESCs attachment/detachment behavior on the thermoresponsive nanobrush surfaces 86
3-3-1 Effect on concentration gradient of ECM grafted in hESCs. 86
3-3-2 Effect of attachemnt/detachment of hESCs on different chain length of PS-b-PEGMA 87
3-4 Intelligent partial detachment of stem cells on thermoresponsive surface 93
3-4-1. Partial detachment of hADSCs on thermoresponsive surface 94
3-4-2. Partial detachment of hESCs on thermoresponsive surface 98
3-5 Characterization of hADSCs and hESCs on thermoresponsive surface 103
3-5-1 Cell viability analysis of hADSCs- LIVE/DEAD assay and MTT assay 103
3-5-2 Immunochemistry analysis of hADSCs 105
3-5-3Characterization of hESCs-Immunochemistry analysis 108
Chapter 4 Conclusions 111
Supplemantal data 113
References 124

參考文獻

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指導教授

樋口亞绀(Akon Higuchi)

審核日期

2015-7-29

推文