人類脂肪幹細胞的膜純化法與分化能力研究

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陳俐伃(Li-Yu Chen) 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

人類脂肪幹細胞的膜純化法與分化能力研究
(Purification and Differentiation of Human Adipose-Derived Stem Cells through Silk Screen/PLGA Hybrid Membranes by Membrane Filtration Method)

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摘要(中)

人類脂肪幹細胞於再生醫學生上具有優勢的幹細胞來源，由於它具有低手術傷害性及可以大量取得等優點。此外，人類脂肪幹細胞具有分化成中胚層細胞的能力，像是骨細胞、軟骨細胞、肌肉細胞等；甚至能分化成外胚層的細胞，像是神經細胞。因此脂肪幹細胞在再生醫學與組織工程學上被視為極具潛力的幹細胞來源。
目前最傳統的組織純化細胞方式為細胞培養法，但這種方法需要長時間的培養與繼代。螢光活性細胞分選 (FACS)和磁力活性細胞分選(MACS)也常選用作為組織細胞的純化萃取，這種利用抗體專一性分離的方法，可能有抗體病毒汙染和高成本的疑慮。而本研究中的膜分離法具有簡單快速等優點，且分離過程在完全無汙染的環境中進行，此項研究試圖從人類脂肪組織中萃取出含有SVF的初代細胞溶液，快速萃取出可表現脂肪幹細胞表面標誌且具有分化能力細胞。
我們使用傾注式的膜過濾法，使用自製的絲綢/聚乳酸-甘醇酸(silk screen/PLGA)合成膜，因絲綢為天然的蛋白質纖維，而聚乳酸-甘醇酸則擁有優良的生物降解性及生物相容性。在初代細胞通過自製的過濾膜後所得之細胞溶液稱之為過濾液，然後再由相反方向注入培養液所得到未能通過膜的細胞之細胞溶液稱之為回收液。本研究中探討初代細胞液的體積及細胞密度的影響，藉由流式細胞儀分析細胞表面標誌和經由誘導分化培養基培養後的基因表現、鹼性磷酸酶活性及Alizarin red & von Kossa staining測試細胞的分化能力，研究結果顯示出18ml細胞密度為500,000 cells/ml的初代細胞溶液，擁有較高的間葉幹細胞表現量及分化能力。由此可以證明人類脂肪幹細胞可以經由膜過濾法(使用自製的膜)被分離在過濾液中。

摘要(英)

Human adipose-derived stem cells (hADSC) represent one of the most promising cell candidates in the field of regenerative medicine, it can easily be extracted in large amount compared to bone marrow stem cells and also exhibits a higher proliferation rate in the medium. Furthermore, hADSCs also have a high differentiation capability into mesoderm (muscle, bone, and cartilage) and ectoderm (nerves and epidermal). The cultivation of adipose tissue cells generates hADSCs with contamination of several other types of cells. Therefore, it is necessary to purify hADSCs before using hADSCs for clinical applications. Purification of hADSCs via the cell culture process requires 5-12 days by using the conventional culture method. In this study, we developed a membrane filtration method to purify hADSCs using novel membranes where the operation time is less than 30 min to purify hADSCs from adipose tissue solution. We prepared silk screen/PLGA hybrid membranes (scaffolds) by a freeze drying method where silk screens (170 mesh size) are natural and readily biodegradable protein fibers to be used for reinforcement of the membranes. PLGA has biodegradability and biocompatibility to be used to generate a sponge pore morphology of the membranes. The average pore size of the silk screen/PLGA hybrid membranes was measured to be 3.5-7µm from scanning electron microscopy. After the preparation of home-made silk screen/PLGA hybrid membranes, we investigated the purification of hADSCs from adipose tissue solution (adipose tissue-derived stromal vascular fraction [SVF]) having different cell density by the membrane filtration method and evaluated the purification efficiency of hADSCs. The mesenchymal stem cell (MSC) markers such as CD44, CD73, and CD90 expressed by hADSCs were less than 10% in the adipose tissue solution (SVF), whereas the MSC markers in the permeate solution were found to be 30-50%, indicating hADSCs were concentrated after permeation through the silk screen/PLGA hybrid membranes, when 9 ml of the adipose tissue solution having 1×106 cells/ml was permeated through the membranes. The MSC markers of the cells after 12 days of culture of the adipose tissue solution (hADSCs purified by the conventional culture method) were found to be 60-80%. The efficiency of hADSC purification in the permeation solution through the silk screen/PLGA hybrid membranes analyzed by MSC markers depended on the cell density of the adipose tissue solution. Currently 1×106 cells/ml was the optimal cell density compared to 2×106 cells/ml or 4×106 cells/ml when 9 ml of adipose tissue solution was used. It was demonstrated that more than two fold higher osteogenic gene expression, Alizarin red staining, and von Kossa staining was observed in the permeate solution compared to the adipose tissue solution (SVF) when the cells were cultured in osteogenic induction medium for 28 days. Therefore, the hADSCs were purified in the permeation solution and demonstrated a superior capacity for osteogenic differentiation than the cells in the adipose tissue solution (SVF). The polyurethane foaming membranes having pore size of 11 µm could not purify hADSCs in the permeate solution. This result indicates that the pore size and membrane material are important factors to purify the hADSCs by the membrane filtration method. It is concluded that the hADSCs can be easily isolated through the permeation through the silk screen/PLGA hybrid membranes, whereas non-hADSCs are blocked by the sieving effect of the membrane pore size and/or adhered on the membranes.

關鍵字(中)

★ 人類脂肪幹細胞
★ 成骨分化
★ 間葉幹細胞
★ 膜純化法

關鍵字(英)

★ Human Adipose-derived Stem Cells
★ Membrane Filtration method
★ Mesenchymal stem cells
★ Osteogenic differentiation

論文目次

Index of Contents
Index of Tables i
Chapter 1 Introduction 1
1-1 Stem Cells 1
1-1-1 Embryonic stem cells (ESCs) 1
1-1-2 Induced pluripotent stem cells (iPSCs) 2
1-1-3 Hematopoietic stem cells (HSCs) 3
1-1-4 Mesenchymal stem cells (MSCs) 3
1-2 Adipose-derived stem cells 4
1-3 Differentiation capacity of adipose-derived stem cells 6
1-3-1 Lineage-specific differentiation potential 6
1-3-2 Adipogenic differentiation 6
1-3-3 Chondrogenic and osteogenic differentiation 7
1-3-4 Myogenic and cardiomyogenic differentiation 8
1-3-5 Other effective factors of MSC and ADSC differentiation 9
1-4 Immunophenotype 10
1-5 Isolation of adipose-derived stem cells 12
1-5-1 Cell isolation 12
1-5-2 Membrane purification method 14
1-6 Flow cytometry 14
1-7 Polymerase chain reaction (PCR) 17
1-7-1 Introduction of PCR 17
1-7-2 The procedure of PCR 18
1-8 Osteogenic differentiation 21
1-8-1 The process of bone development in situ 21
1-8-2 Developmental pathways for bone formation 22
1-8-3 The marker of osteogenic differentiation 26
Chapter 2 Materials and Methods 28
2-1 Materials 28
2-1-1 Chemical 28
2-1-2 Consumable goods 30
2-1-3 Instruments 31
2-2 Experimental method 31
2-2-1 PBS (phosphate buffer saline solution) preparation 31
2-2-2 Culture medium preparation 32
2-2-3 Isolation and culture of adipose tissue-derived stromal cell 32
2-2-4 Culture and passaging of ADSCs 35
2-2-4 Preparation of 1 sheet of silk screen / PLGA hybrid membranes 36
2-2-5 Cell purification (membrane filtration method) 37
2-2-6 Differentiation of adipose tissue-derived stem cell 38
2-2-7 Immunology staining 39
2-2-8 Isolation of total RNA 39
2-2-9 Reverse transcription of mRNA into cDNA 40
2-2-10 PCR (polymerase chain reaction) 42
2-2-11 Alkaline phosphatase activity 44
2-2-12 Alizarin red staining 44
2-2-13 von Kossa staining 45
2-2-14 Quantitative analysis of osteogenesis 45
2-2-15 Scanning electron microscopy (SEM) analysis 46
Chapter 3 Results and Discussion 47
3-1 Characterization of the silk screen/PLGA hybrid membranes 47
3-1-1 SEM observation 47
3-2 Characterization of human adipose derived stem cells (hADSC) 52
3-3 Purification of hADSCs from a primary adipose tissue cell solution using the membrane filtration method 54
3-3-1 Flow cytometry analysis of human adipose tissue solution 55
3-3-1-1 One sheet of silk screen/PLGA hybrid membranes using different cell density and different volume of cell solution 55
3-3-1-2 Permeation through 1, 3, 5 sheet of silk screen/PLGA hybrid membranes using 18 ml cell solution containing 50×104 cells/ml 62
3-4 The ability of adipose tissue cells purified by the membrane filtration method to differentiate into osteoblasts 69
3-4-1 Alkaline phosphatase activity and gene expression 69
3-4-1-1 One sheet of silk screen/PLGA hybrid membranes using different cell density and volume of cell solution 69
3-4-1-2 1, 3, 5 sheets of silk screen/PLGA hybrid membranes using 18 ml of cell solution containing 50×104 cells/ml 74
3-4-2 Alizarin red and von Kossa staining 79
3-4-2-1 One sheet of silk screen/PLGA hybrid membranes using different cell density and volume of cell solution 79
3-4-2-2 hADSC purification through 1, 3, 5 sheet of silk screen/PLGA hybrid membranes using 18 ml cell solution containing 100×104 cells/ml 85
Chapter 4 Conclusion 91
Reference 93

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指導教授

樋口亞绀(Akon Higuchi)

審核日期

2013-7-9

推文