設計應力刺激微流體晶片以探討幹細胞的化學誘導分化之研究

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姓名

程志豪(Jhih-Hao Cheng) 查詢紙本館藏

畢業系所

能源工程研究所

論文名稱

設計應力刺激微流體晶片以探討幹細胞的化學誘導分化之研究

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摘要(中)

由於幹細胞為目前被公認為可以分化成特定細胞的能力，因此在組織工程或再生醫學的應用或研究上逐漸受到重視，但因幹細胞分化容易受到外在環境所影響，而傳統實驗裝置由於對培養環境的控制較差因此在臨床應用或實驗研究稍嫌不足。而近年來發展的微流體晶片系統因其裝置微型化可降低實驗所需的藥劑量且可以模擬體內的微環境、可以進行自動化的控制、搭配即時監控系統、可精準控制的微環境並進行多種刺激等優勢，因此利用在幹細胞的研究上可以說是相當具有潛力。
本研究目的在於設計出可在同一晶片中進行多重應力刺激的微流體晶片，而目前先以設計出可進行多項相同剪應力刺激的微流體晶片裝置，利用流道設計可在實驗中建構多個相似的培養環境並進行相同的剪應力刺激實驗，將培養大鼠骨髓幹細胞(Rat bone marrow stem cells，RM)投以IBMX藥劑讓幹細胞成功分化為神經細胞，也搭配10分鐘不同總流率(0.042 μL/min、1μL/min與15μL/min)含藥與不含藥的流場剪應力刺激，並在刺激後的10個小時內觀察細胞分化情形，發現到有應力刺激的條件下可使細胞的分化時間提前並且也可增加其分化效率，而且於本實驗中也成功製做出可進行正向應力刺激的微流體晶片。

摘要(英)

It is well recognized that stem cells have the ability to genetically differentiate into a specific cell type for promising applications such as tissue engineering or cell therapy. Stem cell is sensitive to the environment during the process of differentiation, so the experimental equipment in traditional that can’t well controlled the cell culture environment isn’t appropriate for stem cells research. Microfluidic techniques have been recently developed for well controlled the cell culture environment. In microfluidic systems, the objective is for these microenvironments to mimic in vivo surroundings. With advantageous characteristics such as optical transparency and the capability for automating protocols, different types of cells can be cultured, screened, and monitored in real time to systematically investigate their morphology and functions under well-controlled microenvironments in response to various stimuli. The microfluidic chips have the great potential in the stem cell research.
The purpose of this study is designing a chip for multiple different stress stimuli microfluidic chip, first we design a chip for multiple same stress stimuli microfluidic chip which is similar to provide multiple culture environment. We also use IBMX induce the RMs differentiate to neuron cell. We also do temporary fluid flow stimulation with or without IBMX under the various rate at 0.042, 1, 15 μL/min, respectively. The results show that fluid flow stimuli not only facilitate RMs to differentiate toward neuronal cells but also shorten the differentiation time. In this study, we also successfully made microfluidic chips for positive stress stimulus.

關鍵字(中)

★ 幹細胞
★ 微流體
★ 物理刺激

關鍵字(英)

論文目次

摘要 ................................................................................................................ I
ABSTRACT ................................................................................................ II
致謝 ............................................................................................................ III
目錄 ............................................................................................................. IV
圖目錄 ...................................................................................................... VIII
表目錄 ......................................................................................................... XI
第一章緒論 ................................................................................................. 1
1.1 前言 ..................................................................................................... 1
1.2 文獻回顧 ............................................................................................. 4
1.3 研究目的 ........................................................................................... 13
1.4 論文架構 ........................................................................................... 14
第二章實驗設計與製作 ........................................................................... 15
2.1 製程設備與耗材 ............................................................................... 15
2.1.1 製程設備 .................................................................................... 15
2.1.2 製程耗材 .................................................................................... 16
2.2 材料選擇 ........................................................................................... 17
2.2.1 SU-8 ............................................................................................ 17
2.2.2 聚二甲基矽氧烷(PDMS) .......................................................... 18
2.3 黃光微影製程 ................................................................................... 19
2.3.1光罩製作 ..................................................................................... 20
2.3.2晶圓清潔(wafer cleaning).......................................................... 22
2.3.3光阻塗佈(spin coating) .............................................................. 23
2.3.4 軟烤(soft bake) .......................................................................... 24
2.3.5 曝光(exposure) .......................................................................... 25
2.3.6 曝後烤(post exposure bake) ...................................................... 25
2.3.7 顯影(develop) ............................................................................. 25
2.3.8 硬烤(hard bake) ......................................................................... 26
2.4 晶片製作 ........................................................................................... 26
2.4.1翻膜 ............................................................................................. 27
2.4.2氧電漿接合 ................................................................................. 28
2.5 系統架設 ........................................................................................... 30
2.5.1微流體晶片 ................................................................................. 31
2.5.2系統環境控制 ............................................................................. 31
2.5.3光學觀測元件 ............................................................................. 32
第三章實驗方法 ....................................................................................... 33
3.1 藥品配製 ........................................................................................... 34
3.2 細胞培養方法 ................................................................................... 36
3.3 實驗操作流程 ................................................................................... 41
3.3.1實驗前準備 ................................................................................. 41
3.3.2 細胞植入晶片 ............................................................................ 42
3.3.3 藥物誘導分化 ............................................................................ 44
3.3.4 染色步驟 .................................................................................... 45
3.4細胞影像分析 ................................................................................... 50
3.4.1細胞數量分析 ............................................................................. 50
3.4.2分化率的分析 ............................................................................. 50
第四章實驗結果與討論 ........................................................................... 51
4.1晶片內各培養區的細胞均勻度 ....................................................... 51
4.2 藥物與流場剪力對分化結果之影響 .............................................. 56
4.2.1藥物濃度測試 ............................................................................. 56
4.2.2藥物與流場剪應力的影響 ......................................................... 59
4.3不同流道寬度的陣列式晶片 ........................................................... 71
第五章正向應力刺激的微流體晶片 ...................................................... 74
5.1流道設計 ........................................................................................... 74
5.2晶片製作 ........................................................................................... 76
VII
5.3系統架設 ........................................................................................... 78
第六章結論與未來展望 ........................................................................... 80
6.1結論 ................................................................................................... 80
6.2未來展望 ........................................................................................... 81
文獻回顧 ..................................................................................................... 83
附錄A ......................................................................................................... 88

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65. ROHM&HAAS, MEGAPOSIT SPR220 SERIES PHOTORESISTS

指導教授

曹嘉文(Chia-Wen Tsao)

審核日期

2013-7-27

推文