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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題名:	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
作者:	陳俐伃;Chen,Li-Yu
貢獻者:	化學工程與材料工程學系
關鍵詞:	人類脂肪幹細胞;成骨分化;間葉幹細胞;膜純化法;Human Adipose-derived Stem Cells;Membrane Filtration method;Mesenchymal stem cells;Osteogenic differentiation
日期:	2013-07-09
上傳時間:	2013-08-22 11:40:13 (UTC+8)
出版者:	國立中央大學
摘要:	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?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。 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 3.5-7?m 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 ?m 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.
顯示於類別:	[化學工程與材料工程研究所] 博碩士論文

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