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姓名	楊琇婷(Siou-Ting Yang)  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	膜純化法及免疫抗體磁珠法用於分離及體外增殖血液幹細胞之研究 (Separation and ex vivo expansion of hematopoietic stem cells from human blood by membrane filtration method and magnetic associated sorting method)
相關論文	★ 於不同彈性係數的生醫材料上體外培植造血幹細胞 ★ 藉由調整水凝膠之表面電荷及軟硬度並嫁接玻連蛋白用以培養人類多功能幹細胞 ★ 可見光對羊水間葉幹細胞成骨分化之影響 ★ 可見光調控神經細胞之基因表現及突觸生長 ★ 人類表皮成長因子的結構穩定性及生物活性測定 ★ 微環境對羊水間葉幹細胞多功能性基因表現及分化之影響 ★ 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 ★ 小鼠脂肪幹細胞之膜純化法及細胞外間質對人類脂肪幹細胞影響之研究 ★ 利用具有奈米片段與細胞外間質蛋白質的表面改殖材質進行臍帶血造血幹細胞體外培養 ★ 在不同培養條件下針對大腸癌細胞及組織中癌細胞進行純化、剔除及鑑定之研究 ★ 羊水間葉幹細胞培養於細胞外間質改質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 ★ 具有抗藥性之大腸癌細胞株能提高癌胚抗原的表現，但並非是癌症起始細胞 ★ 羊水間葉幹細胞培養於接枝細胞外間質寡肽與環狀肽具有最佳表面硬度的生醫材料,其增殖能力及多能性之研究 ★ 人類體細胞從組成誘導型多能性幹細胞培養在無飼養層上 ★ 使用不同孔洞大小之耐倫薄膜從脂肪組織中分離及純化人類脂肪幹細胞之研究
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摘要(中)	現雖有不同幹細胞分離技術，其中薄膜分離技術是由本實驗室提出且為最簡便、易保持無菌、及維持細胞活性的方法，為臨床醫師認為最具潛力的細胞分離術。臍帶血之血液幹細胞經由膜分離技術，可得到7-20%的回收率。此研究也發展臍帶血幹細胞之體外直接膜培養，可得到6.6倍的血液幹細胞增殖倍數。為了最終應用於臨床醫療之目的，此研究更進ㄧ步設計閉鎖式循環膜培養系統，封閉的培養環境可降低污染的可能性，而不斷循環的培養基則可提供充足的養分給予附著在膜上的血液幹細胞，利用此ㄧ系統可得到14.6倍的血液幹細胞增殖率。除了創新的膜分離培養技術外，此研究也比較了目前普遍使用的免疫磁珠法分離血液幹細胞，被分離的血液幹細胞進而培養在不同的二維及三維培養材料上，由此研究結果發現，血液幹細胞培養在三維材料上，其增殖率小於二維材料，這可能由於血液幹細胞表面表現黏著的分子(CD164)，容易附著於3D膜的表面之緣故。此研究也進行了血液幹細胞的分化能力鑑定。閉鎖式循環膜培養系統與免疫磁珠法分離的血液幹細胞培養可得到接近的血液幹細胞增殖倍率，但閉鎖式循環膜培養系統從分離到進行培養只需30分中的操作時間，免疫磁珠法卻需耗費5-8個小時，閉鎖式循環膜培養系統為一具有潛力之血液幹細胞體外增殖的方法。
摘要(英)	Stem cell separation through membrane filtration method is simple and inexpensive, and sterility is easy to maintain during the process. The recovery ratio of HSCs from umbilical cord blood (UCB) was found to be 7-20% depending on the recovery solution. A high recovery ratio of HSCs was obtained when 20 wt% of dextran was used as the recovery solution. Direct ex vivo expansion of hematopoietic stem cells (HSCs) from UCB by membrane filtration method has been developed in this study. This study also reports the expansion ratio and ability of colony-forming units of HSCs from UCB purified by conventional Ficoll-Paque and MACS medthod, batch type method of direct ex vivo expansion through membranes and bioreactor of perfusion method of direct ex vivo expansion through membranes, and compared these results.　Ex vivo expansion of HSCs compared to initial number of HSCs in UCB was found to be 6.6 folds when was used as a rinsing solution in batch type of direct ex vivo expansion of HSCs through the PU-COOH membranes. Ex vivo expansion of HSCs in 3D culture was found to be worse than that in 2D culture using HSCs purified by Ficoll-Paque and MACS method. This is because pluripotent HSCs adhered on PU membranes tightly, and HSCs in PU membranes of 3D culture could not be detached even after pipetting with washing solution. However, ex vivo expansion of HSCs in bioreactor of perfusion method was found to be similar ex vivo expansion fold compared to that in 2D culture using HSCs purified by conventional Ficoll-Paque and MACS method, which needs longer working time (e.g., 5 hours). The bioassay of HSCs by colony-forming units where HSCs were cultured after purification of HSCs from UCB by conventional Ficoll-Paque and MACS method and by our direct ex vivo expansion method was also investigated. Perfusion type of direct ex vivo expansion method of HSCs from UCB through membranes can provide high ex vivo expansion fold with simple procedure and short working time for HSC purification before HSC culture.
關鍵字(中)	★ 血液幹細胞 ★ 體外增殖 ★ 幹細胞純化	關鍵字(英)	★ ex vivo expansion ★ hematopoietic stem cells ★ stem cell purification
論文目次	中文摘要 I ABSTRACT II 誌謝 III ACKNOWLEDGEMENT IV INDEX of CONTENTS V INDEX of FIGURES VIII INDEX of TABLES XII CHAPTER ONE : INTRODUCTION 1 1-1 Hematopoietic Stem Cell 1 1-1-1 Types of HSCs 1 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) 5 1-2-2 Magnetic-Activated Cell Sorting (MACS) 8 1-2-3 Membrane Purification 10 1-3 Ex Vivo Expansion of HSCs 10 1-3-1 Culture Medium Effect of Ex Vivo Expansion 12 1-3-2 Culture Material Effect of Ex Vivo Expansion 13 1-3-2-1 Conventional synthetic and natural polymeric materials 15 1-3-2-2 Materials manufactured with nanotechnology and having nanosegments 19 1-3-2-3 Polymeric materials modified with immobilized proteins and oligopeptides 23 1-3-2-4 Polymeric materials modified with immobilized glycosaminoglycans 29 1-3-2-5 Materials for 3D culture 31 1-3-3 Bioreactor for HSCs 34 1-3-3-1 Bioreactor design and operation 34 1-3-3-2 Perfusion chambers 34 1-3-3-3 Stirred reactors 35 1-3-3-4 Packed and fluidized bed reactors 35 1-3-3-5 Other reactor types 36 1-4 HSCs Analysis 36 1-4-1 Flow Cytometry Analysis 36 1-4-1-1 The Use of the CD34 Surface Marker to Characterize Primitive Cells 37 1-4-1-2 Flow Cytometry Protocols For the Enumeration of CD34+ cells 38 1-4-2 Colony Forming Cell (CFC) Assay 40 CHAPTER TWO : EXPERIMENT AND METHOD 42 2-1 Materials 42 2-1-1 Preparation of surface-modified PU foaming membranes and surface-modified polystyrene dishes 43 2-1-2 Platelet Poor Plasma (Plasma A) 46 2-1-3 Buffer solution 46 2-2 HSCs Purification 47 2-2-1 Membrane Purification 47 2-2-2 Magnetic Cell Sorting (MACS) 49 2-2-2-1 Preparation of cord blood cells 49 2-2-2-2 Magnetic labeling and magnetic separation of CD34+ cells 49 2-3 HSCs Ex Vivo Expansion 52 2-3-1 Direct Ex Vivo Expansion 52 2-3-1-1 Recovery Solution Direct Ex Vivo Expansion 52 2-3-1-2 Direct Ex Vivo Expansion on Membranes 52 2-3-2 Ex Vivo Expansion of HSCs after MACS Purification 53 2-3-3 Perfusion Cultivation of HSCs 53 2-4 HSCs Analysis 56 2-4-1 Flow Cytometry Analysis 56 2-4-2 Colony Forming Cell (CFC) Assay 56 CHAPTER THREE : RESULTS & DISCUSSION 58 3-1 HSCs Purification by Membrane Purification Method 58 3-1-1 Peripheral Blood Permeation through 5 μm PU-COOH Membranes Using Different Recovery Solutions 58 3-1-1-1 Permeation of the peripheral blood cells 58 3-1-1-2 Recovery of the peripheral blood cells by different recovery solutions 58 3-1-2 Permeation of UCB through PU-COOH Membranes Having Different Pore Size Using Several Recovery Solutions 61 3-1-2-1 Permeation of UCB cells through membranes having different pore size 61 3-1-2-2 Effect of recovery solution on recovery ratio of UCB cells through membranes having different pore size 62 3-2 Ex Vivo Expansion of HSCs 69 3-2-1 Direct Ex Vivo Expansion of HSCs from UCB 69 3-2-2 Ex Vivo Expansion of Cells after HSC Purification by Ficoll-Paque and MACS Methods from UCB 75 3-2-3 CFU assay of Ex Vivo Expanded HSCs after HSC Purification by Ficoll-Paque and MACS Methods from UCB 83 3-2-4 Bioreactor for HSCs 92 CHAPTER FOUR : CONCLUSION 95 REFERENCE 98
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指導教授	樋口亞紺(Akon Higuchi)	審核日期	2009-7-10
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