設計微流體晶片應用於人體胎盤幹細胞的物理/化學誘導分化之研究

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姓名

姜孟志(Meng-zhi Chiang) 查詢紙本館藏

畢業系所

機械工程學系

論文名稱

設計微流體晶片應用於人體胎盤幹細胞的物理/化學誘導分化之研究
(Applying micro chip system in mechanical and chemical inductions of human PDMCs differentiation)

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摘要(中)

幹細胞本身具有良好的自我再生特性與分化成不同細胞的能力，因此在再生醫學的應用上逐漸受到重視，但也因為幹細胞的分化容易受到環境因子的影響，使得幹細胞在臨床應用上仍然面臨嚴峻的挑戰。利用微機電製程技術所設計的微流體晶片系統能夠提供給幹細胞一個更加精確的控制且接近人體體內尺度(In vivo-like)的培養環境，此外，微流體晶片還具有體積小、減少汙染、降低樣本耗費與試劑成本以及可在螢光顯微鏡下進行即時觀察等特點。
本研究的目的是在建立起一個新型的微流體培養平台提供給胎盤幹細胞(PDMCs)使用，讓胎盤幹細胞可以穩定地進行長時間的培養與分化。實驗結果證實此裝置可滿足生物相容性之需求，細胞可以在連續灌注的培養條件下進行超過10天的長期培養。另外，本研究也利用藥物IBMX讓胎盤幹細胞分化成神經細胞，並於投藥過程的前10分鐘給予三組不同注入流率(0.083 μL/min、2 μL/min與30 μL/min)之流場刺激，並觀察在刺激之後的72小時內其對細胞分化之影響。實驗結果顯示較高的流率下具有增進細胞提早分化的發生。

摘要(英)

Stem-cell biology for the applications of regenerative medicine is gathering great interests because of their self-renewal property and ability to differentiate into many types of cells. Since differentiation of stem cells is sensitive to environmental factors, stem cells for clinical use are encountering a vast challenge. The microfluidic chip system fabricated by micro electro mechanical systems (MEMS) technology is able to provide microenvironments which can mimic in vivo surroundings and to be well-controlled. Besides, these devices are characterized as a smaller size, less sample/reagent consumption, reduced risk of contamination, and real-time optical analysis.
In this study, a new microfluidic chip system is developed which can culture and differentiate PDMCs in in vivo-like microenvironments. Experimental results indicate that the microfluidic chip system can achieve a well biocompatibility, and the cells can be cultured under perfusion in the chips over 10 days. We also used IBMX to induce the differentiation of PDMCs into neuron within temporary fluid flow stimuli under the various rate at 0.083, 2 and 30 μL/min, respectively. The results illustrate that the fluid flow could promote cells differentiation earlier at higher flow rates.

關鍵字(中)

★ 灌注式培養
★ 分化
★ 幹細胞
★ 微流體
★ 微機電製程

關鍵字(英)

★ differentiation
★ stem cell
★ microfluidic
★ perfusion culture
★ MEMS

論文目次

摘要 I
ABSTRACT . II
誌謝 III
目錄 IV
圖目錄 . VIII
表目錄 XII
第一章緒論 1
1.1 前言 . 1
1.2微機電系統與微流體晶片 . 2
1.3文獻回顧 4
1.4研究動機 . 10
1.5論文架構 . 11
第二章設計與製程 12
2.1 製程設備與耗材 . 12
2.1.1 製程設備與實驗儀器 12
2.1.2 製程材料與實驗耗材 13
2.2 材料選擇 13
2.2.1 SU-8 13
2.2.2 聚二甲基矽氧烷 15
2.3 黃光製程 16
2.3.1 光罩製作 17
2.3.2 晶圓清潔(Wafer cleaning) . 18
2.3.3 塗佈光阻(Spin coating) 19
2.3.4 軟烤(Soft bake) 21
2.3.5 曝光(Exposure) 22
2.3.6 曝後烤(Post exposure bake) 22
2.3.7 顯影(Develop) 23
2.3.8 硬烤(Hard bake) 23
2.4 翻模過程 24
2.5 脫模與CONNECTOR製作 24
2.6 氧電漿接合 25
2.7 實驗系統架設 29
2.7.1 微流體晶片 . 29
2.7.2 系統環境控制元件 30
2.7.3 光學觀測裝置 . 30
第三章實驗方法 32
3.1 實驗儀器與藥品 . 32
3.1.1 使用儀器 32
3.1.2 實驗藥品 33
3.2 藥品配製 34
3.3 細胞來源與培養方法 37
3.4 實驗操作流程 41
3.4.1 實驗前的準備 . 42
3.4.2 細胞的種植 . 46
3.4.3 藥物誘導分化 . 47
3.4.4 染色步驟 49
3.5 細胞影像處理與分析 55
3.5.1 細胞數量測定 . 55
3.5.2 分化率的分析 . 55
第四章實驗結果與討論 56
4.1 微晶片系統的培養 56
4.2結合藥物與流場之分化結果 62
4.2.1 流率0.083 μL/min 62
4.2.2 流率2 μL/min 66
4.2.3 流率30 μL/min 69
4.3 實驗結果討論 75
第五章結論與未來展望 80
5.1 結論 . 80
5.2 未來展望 81
參考文獻 . 83

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指導教授

曹嘉文(Chia-wen Tsao)

審核日期

2012-1-19

推文