博碩士論文 973204030 詳細資訊




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姓名 李珮慈(Pei-Tsz Li)  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 奈米片段與細胞外基質之改質膜用於臍帶血中造血幹細胞之純化與培養
(Purification and Cultivation of Hematopoietic Stem Cells through Surface-modified Membranes having Nano-segments and Extracellular Matrix Proteins from Umbilical Cord Blood)
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摘要(中) 將血液細胞、幹細胞和特殊組織細胞有效而簡易地分離是一項重要的課題。此研究中,我們使用經過表面改質的多孔性高分子膜來分離造血幹細胞。臍帶血經由過濾的方式通過高分子膜,隨即以反方向再一次過濾沖洗液,接著將留有造血幹細胞的膜直接置入培養基裡,使幹細胞在三維的培養環境中增殖。負電位的造血幹細胞和帶有羧基的膜之間產生輕微的靜電排斥作用而形成溫和的交互作用,經過培養後,最高可以得到6.6倍的增殖數量。接著我們使用一般常見的免疫磁珠分離法來純化造血幹細胞,培養於接上奈米片段或細胞外基質(Fibronectin, Collagen, Matrigel, Laminin)的二維表面,與高分子膜的三維環境做比較。相較於臍帶血中原有的造血幹細胞數量,培養在接上胜肽(CS-1)的平面可以得到16.5倍的增殖。
總結以上,臍帶血中的造血幹細胞經過膜過濾分離後,直接進行體外培養的實驗步驟相當簡易,在無菌狀態下只需要三十分鐘以內的操作時間,即可以成功地增加造血幹細胞數量。在造血幹細胞的分化能力測試中,增殖後的細胞仍保留良好的造血能力。然而,常應用在二維培養的免疫磁珠分離法,則需要五到八小時的操作時間。我們利用直接膜培養的概念設計出閉鎖式循環系統,可以呈現類似骨髓的環境,此方法可以有效地將體外培養後的造血幹細胞應用於臨床實驗。
摘要(英) Efficient and simple cell separation is an important issue for the isolation of blood cells, stem cells and specific tissue cells. Separation of hematopoietic stem cells (HSCs, CD34+ cells) was investigated using surface-modified polyurethane (PU) foaming membranes in this study. HSCs were collected from umbilical cord blood permeated through the membranes, and subsequently the rinsing solution permeated through the membranes. Following filtration of rinsing solution, the membranes were inserted into culture medium where the HSCs could be expanded in a three-dimensional (3D) environment ex vivo. The highest ex vivo expansion of HSCs (i.e. 6.6) was obtained using PU membranes having carboxylic groups (PU-COOH). The carboxylic acid group can generate mild interaction between HSCs and the membranes by electrostatic repulsion because of negatively charged surface of the cells, which leads to efficient recovery of HSCs due to easy detachment of HSCs from the surface of the membranes. We further investigated HSC cultivation on three dimensional (PU-membrane) and two dimensional dishes immobilized nano-segments and extracellular matrix protein (e.g., Fibronectin, Collagen, Matrigel, and Laminin). Ficoll-Paque and MACS (Magnetic associated cell sorting) methods are typically employed for HSC purification. 16.5 times as much as the number of HSCs in original UCB was expanded on CS-1-immobilized dishes.
In summary, the direct ex vivo expansion of HSCs through membranes provides successful ex vivo expansion of HSCs which possess excellent hematopoietic ability in colony-forming unit assays (i.e., more pluripotent colony numbers were identified). It is a simple procedure that can be performed under sterile conditions in short working time (less than 30 min) before HSC culture. In comparison, the 2D cultures using HSCs purified by the Ficoll-Paque and MACS method requires 5-8 hr of operation time. The direct ex vivo expansion of HSCs from UCB by the perfusion method is mimicking the bone marrow niche and should be effective for the clinical application of the ex vivo expansion of HSCs.
關鍵字(中) ★ 體外增殖
★ 培養材料
★ 細胞外基質
★ 造血幹細胞
關鍵字(英) ★ extracellular matrix
★ culture materials
★ hematopoietic stem cells
★ ex vivo expansion
論文目次 中文摘要 i
ABSTRACT ii
INDEX OF CONTENTS iii
INDEX OF FIGURES vi
INDEX OF TABLES x
INDEX OF ABBREVIATION xi
CHAPTER ONE: INTRODUCTION 1
1-1 Hematopoietic Stem Cell 1
1-1-1 Types of HSCs 2
1-1-2 Molecular and cellular mechanisms of hematopoiesis 2
1-2 Purification of Hematopoietic Stem Cell 5
1-2-1 Fluorescence-Activated Cell Sorting (FACS) 6
1-2-2 Magnetic-Activated Cell Sorting (MACS) 8
1-2-3 Membrane Purification 10
1-3 Ex Vivo Expansion of HSCs 11
1-3-1 Culture Medium Effect of Ex Vivo Expansion 12
1-3-2 Culture Material Effect of Ex Vivo Expansion 14
1-3-2-1 Conventional synthetic and natural polymeric materials 15
1-3-2-2 Materials manufactured with nanotechnology and having nanosegments 20
1-3-2-3 Polymeric materials modified with immobilized proteins and oligopeptides 23
1-3-2-4 Polymeric materials modified with immobilized glycosaminoglycans 33
1-3-2-5 Materials for 3D culture 35
1-3-3 Bioreactor for HSCs 38
1-3-3-1 Bioreactor design and operation 38
1-3-3-2 Perfusion chambers 38
1-3-3-3 Stirred reactors 39
1-3-3-4 Packed and fluidized bed reactors 39
1-3-3-5 Other reactor types 40
1-4 HSCs Analysis 40
1-4-1 Flow Cytometry Analysis 40
1-4-1-1 The Use of the CD34 Surface Marker to Characterize Primitive Cells 41
1-4-1-2 Flow Cytometry Protocols For the Enumeration of CD34+ cells 42
1-4-2 Colony Forming Cell (CFC) Assay 44
CHAPTER TWO: EXPERIMENTAL AND METHOD 46
2-1 Materials 46
2-1-1 Preparation of Surface-Modified PU Foaming Membranes and Surface-Modified Polystyrene Dishes 47
2-1-2 Preparation of PS-NH2 dishes coated and immobilized with extracellular proteins. 50
2-1-3 Umbilical Cord Blood (UCB) and Platelet Por Plasma (Plasma A) 51
2-1-4 Buffer solution 52
2-2 Characterization Analysis 52
2-2-1 Scanning electron microscopy (SEM) analysis 52
2-2-2 X-ray photoelectron spectra (XPS) 53
2-2-3 Quantification of reactive solid-supported primary amino group 53
2-2-3-1 Activation using ITL solution 53
2-2-3-2 Reaction with Micro BCA™ working solution 53
2-3 HSCs Purification 54
2-3-1 Membrane Purification method 54
2-3-2 Magnetic Cell Sorting (MACS) method 56
2-3-2-1 Preparation of cord blood cells 56
2-3-2-2 Magnetic labeling and magnetic separation of CD34+ cells 57
2-4 Ex Vivo Expansion of HSCs 60
2-4-1 Direct Ex Vivo Expansion of HSCs on Membranes 60
2-4-2 Ex vivo Expansion of HSCs on Surface-modified Dishes (2D Culture) 60
2-4-3 Perfusion Cultivation of HSCs 61
2-5 HSCs Analysis 63
2-5-1 Flow Cytometry Analysis 63
2-5-2 Colony Forming Cell (CFC) Assay 63
CHAPTER THREE: RESULTS AND DISCUSSION 65
3-1 Characterization analysis of the surface of culture dishes 65
3-2 HSCs Purification from UCB by membrane filtration method 71
3-2-1 Effect of recovery solution using different volume of UCB permeated through PU-COOH membranes having pore size = 6 μm 71
3-2-2 Effect of membrane having different functional groups using 1 mL of UCB for permeation and PPP as recovery solution 77
3-3 Ex Vivo Expansion of HSCs from UCB 80
3-3-1 Direct Ex vivo Expansion of HSCs on Membranes 80
3-3-2 Ex vivo Expansion of HSCs on Surface-modified Dishes (2D Culture) 85
3-3-3 CFU assay of HSCs after ex vivo expansion 97
CHAPTER FOUR: CONCLUSION 108
REFERENCE 111
APPENDIX 128
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指導教授 樋口亞紺(Higuchi Akon) 審核日期 2010-7-15
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