博碩士論文 107324022 詳細資訊




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姓名 劉雅筑(Ya-Chu Liu)  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 設計連續培養系統應用於人類多能性幹細胞之增生於熱敏性高分子塗佈基材表面
(Design of Continuous Culture of Human Pluripotent Stem Cells on Thermoresponsive Polymeric Surface)
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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,在臨床的醫學治療研究上具有極大的淺力。然而,臨床治療需要極大量的人類多性幹細胞,而一般的細胞培養採用的是費時又費力的批次培養。為解決上述問題,本實驗室利用熱敏感材料設計一個連續是細胞培養。將人類胚胎幹細胞培養於熱敏性基材表面,當我們需要收集細胞時,僅需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)) 以改變基材表面之親疏水性質以達到細胞部分脫附效果。而未脫附細胞將繼續培養於溫度高於低臨界溶解溫度的原熱敏性基材上。
因人類多能幹細胞必須貼附於特定的細胞外基質(Extracellular matrix (ECM))表面上,然而在此研究中,細胞外基質會隨著每一次收集細胞的次數而跟著減少;因此,在此實驗中,為確保細胞維持一定的生長效率,必須再加入一些ECM以確保,細胞能貼附在熱敏感材料,此方法不同於一般的ECM塗佈方法(pre-coating way),其必須既能同時培養細胞,又能同時加入ECM,我們稱此方法為非塗佈法(un-coating way).
此研究選用重組玻連蛋白(recombinant vitronectin(rVN))及重組層粘連蛋白(recombinant laminin-511(iMatrix-511))兩種細胞外基質。研究在非塗佈且不同比例混合之兩種ECM的環境下,對於細胞生長之影響,並找出最適當的混合比例濃度,再將此非塗佈方法及條件應用於連續式的培養。
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)),培養於非塗佈(un-coating way)的環境及兩種ECM適合的混合比例下長期培養,仍保有原有的多功能性以及分化能力。此非塗佈的方法能避免在塗佈時所使用的溶劑,影響未能自熱敏感材料上脫附的細胞死亡。若此連續培養方式從2D培養方式到3D培養系統,將成為醫學應用領域的一大益處。
摘要(英) Stem cells are attractive source for tissue engineering applied on regenerative medicine, translational medicine, and drug discovery. Batch type culture is the typical process for stem cell culture, which is laborious and expansive. Moreover, the digestive enzymes or EDTA solutions to detach stem cells are causing cell damage and increasing production costs. For reducing the costs and laborious processes, I developed the continuous culture system culturing the human pluripotent stem cells (hPSCs) on thermoresponsive polymer surfaces. hPSCs could be detached from the thermoresponsive polymer surfaces by reducing the medium temperature below the lower critical solution temperature (LCST) of the thermoresponsive polymer. After reducing the temperature, the thermoresponsive surface becomes more hydrophilic, creating an unfavorable environment for cell attachment. Subsequently, hPSCs can be partially detached by gentle pipetting. The remained cells could be cultivated and would be confluent again by adding fresh medium. This continuous culture system could be used for 3D-cultivation. However, the barrier of applying this system is that hPSCs became harder to attach on the thermoresponsive surfaces coated with the extracellular matrix (ECM) after multicycles. This should be caused by degradation of ECM after long-term culture. Therefore, I investigated the precoated manner and uncoated manner with different ratios of ECMs; recombinant vitronectin (rVN) and recombinant laminin-511 (iMatrix-511). Comparing with traditional precoated process, the uncoated method where optimal ratio and concentration of ECMs were added into culture medium upon seeding cells, provided cost-effective and time-efficient method and maintained hPSCs pluripotency during long-term culture. This uncoated method that was applied to the continuous culture system would save time cost and accelerate the development of regenerative medicine.
關鍵字(中) ★ 熱敏感材料
★ 人類多能幹細胞
★ 連續培養
關鍵字(英) ★ thermoresponsive surface
★ human pluripotent stem cell
★ continuous cell cultivation
論文目次 Abstract II
致謝 III
Index of Content IV
Chapter 1. Introduction 1
1-1 Stem cells 1
1-1-1 Human pluripotent stem cells 2
1-1-1-1 Human embryonic stem cells 3
1-1-1-2 Induced human pluripotent stem cells 4
1-2 Two-dimensional (2D) culture of hPSCs 6
1-2-1 Human pluripotent stem cell (hPSC) culture on Matrigel 7
1-2-2 hPSC culture on ECM-immobilized surfaces 7
1-3 Physical cues effect of cell culture biomaterials on hPSC fate 11
1-3-1 The effect of biomaterial elasticity on hPSC fate 12
1-3-2 The effect of biomaterial hydrophilicity on hPSC fate 13
1-4 Continuous hPSCs cultivation on thermoresponsive surface 14
1-4-1 Characteristics of thermoresponsive Poly (N-isopropylacrylamide) 15
1-4-2 hPSCs culture on thermoresposive surface 17
1-4-3 Cell sheets prepared on thermoresponsive surface 20
1-5 Characterization of human Pluripotent Stem Cells (hPSCs) 22
1-5-1 Cell morphology of hPSCs 24
1-5-2 Pluripotent Gene Expression 24
1-5-3 Differentiation ability 26
1-6 Goal of this study 28
Chapter 2. Materials and Methods 30
2-1 Materials 30
2-1-1 Cell lines 30
2-1-2 Cell culture media, buffer and others 30
2-1-3 Cell culture substrates 31
2-1-4 Immunostaining analysis 32
2-2 Methods 34
2-2-1 Pre-coating and un-coating comparison on TCPS 34
2-2-2 Preparation of Thermoresposive Dishes 35
2-2-3 hPSCs Culture on the Thermoresponsive Dishes 37
2-2-3-1 hPS Cell Seeding 37
2-2-3-2 hPSCs Detached from Thermoresponsive Surface by Reducing Temperature 38
2-2-4 Characterization of hPSC 39
2-2-4-1 Cell Density Measurement 39
2-2-4-2 Differentiation Rate of hPSCs Colonies 41
2-2-4-3 Expansion Fold Change of hPSCs 41
2-2-4-4 Immunofluorescence Staining 42
2-2-4-5 Embryo Body (EB) Formation in Vitro Assay 45
2-2-4-6 hPSCs Differentiation into Cardiomyocytes 45
2-2-5 Characterization of Thermoresposive Surface 47
2-2-5-1 Contact Angle (CA) measurements 47
2-2-5-2 X-ray Photoelectron Spectroscopy (XPS) measurements 47
2-2-5-3 Fourier-Transform Infrared Spectroscopy (FTIR) measurements 48
Chapter 3. Results and Discussion 50
3-1 Characterization of the thermoresponsive surface 50
3-1-1 X-ray photoelectron spectroscopy (XPS) analysis for pre-coating method and un-coating method on TCPS 50
3-1-2 X-ray photoelectron spectroscopy (XPS) analysis for pre-coating method and un-coating method on thermoresponsive surface 54
3-1-3 Water Contact Angle (CA) Analysis 57
3-2 Pre-coating and Un-coating Methods of hPSCs Culture 60
3-2-1 Human PSC Culture on Several ECM-coated Surface 60
3-2-2 Human iPSC Culture by Un-coating Method with Different Ratio of Mixed ECMs 65
3-2-3 Universal hiPSCs and hESCs Culture Using Mixed ECMs 71
3-3 Characterization of hPSCs after long-term cultivation 76
3-3-1 Immunostaining Analysis on Pluripotency of hPSCs After Long-term Cultivation 76
3-3-2 Immunostaining Analysis on Differentiation Ability of hPSCs in Vitro After Long-term Cultivation 78
3-3-3 Cardiomyocyte Differentiation of hiPSC using Mixed ECMs 81
3-3-4 Flow Cytometry 84
3-4 Continuous Culture of hPSCs on the Thermoresponsive Surfaces 85
3-4-1 The Optimal Concentration of Mixed rVN and iMatrix-511 for Thermal Detachment of hPSCs 85
3-4-2 Continuous cultivation of hiPSCs on the thermoresponsive surfaces 90
Chapter 4. Conclusions 95
Reference 97
Supplementary Data 108
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指導教授 樋口亞紺(Akon Higuchi) 審核日期 2020-8-20
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