濃度梯度微晶片製作分析及細胞運動檢測應用｜國立中央大學博碩士論文系統

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研究生：	周郁舜 Yu-shun Chou
論文名稱：	濃度梯度微晶片製作分析及細胞運動檢測應用 Microfluidic chemotaxis device for measuring the cell migration of the placenta-derived multipotent cells
指導教授：	鍾志昂 Chin-ang Chung
口試委員:
學位類別：	碩士 Master
系所名稱：	工學院 - 機械工程學系 Department of Mechanical Engineering
論文出版年：	2013
畢業學年度：	101
語文別：	中文
論文頁數：	110
中文關鍵詞：	微流體晶片、人類胎盤源多功能幹細胞、第一型膠原蛋白、濃度梯度分佈、化學趨向性
外文關鍵詞：	microfluidic device, placenta-derived multipotent cell, type I collagen, concentration gradient, chemotaxis
相關次數：	點閱：13 下載：0
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由於微流體晶片具有降低成本、提升分析效率與可進行即時觀察等優點，對於生醫科技來說擁有相當大的發展潛力，因此近年來許多學者將微流體裝置應用到生醫領域方面的研究。本文設計製作一個微流體裝置，讓人類胎盤源多功能幹細胞在其系統區域內進行正常的生長與運動，而且此微流體晶片可以產生類似線性分佈的濃度梯度。此外藉由實驗來觀察人類胎盤源多功能幹細胞於第一型膠原蛋白濃度梯度分佈下其運動之行為，且膠原蛋白濃度梯度的初始範圍為0-3μM，根據實驗結果顯示在注入流率為1 μL/min下，人類胎盤源多功能幹細胞無法進行正常的生長與運動，至於注入流率為0.05 μL/min時，人類胎盤源多功能幹細胞能夠正常的生長並進行遷移的行為，但是並沒有明顯的化學趨向性反應。

Recently, microfluidic device has been applied to the biological and medical fields because of its huge potential for high throughput screening and advantages such as experiment cost down, increasing analysis efficiency and real time observation.This study aimed to create a microfluidic concentration generator for measuring the chemotactic migration of the placenta- derived multipotent cells (PDMCs) responding to type I collagen. The microfluidic device could generate linear-like concentration gradients by using cascade branches of micro-channels.
The collagen solution was pumped in concentration generator at two flow rates of 1 μL/min and 0.05 μL/min respectively to establish the type I collagen gradients. The type I collagen concentration gradient ranged between 0 and 3.03 μM. According to the experimental results, the PDMCs scarcely migrated and were washed off by the flow at 1 μL/min. The PDMCs showed random walks when the flow rate was 0.05 μL/min. However, the PDMCs showed no directional migration in response to the collagen gradient. This might be because the concentration of 3μM had saturated the chemotactic behavior of the cells.

摘要 ............................................................................................................................ I
Abstract ................................................................................................................... II
致謝 ......................................................................................................................... III
目錄 ......................................................................................................................... IV
表目錄 ..................................................................................................................... VI
圖目錄 .................................................................................................................... VII
第一章 緒論.............................................................................................................. 1
1 前言 ........................................................................................................ 1
2 微機電系統與微流體晶片 ...................................................................... 3
3 文獻回顧 ................................................................................................. 5
3.1 細胞運動 ...................................................................................... 5
3.2 微流體晶片進行化學趨向性實驗 ............................................... 7
4 研究目的 ................................................................................................. 9
第二章 微晶片設計與製程 .....................................................................................13
1 製程設備 ................................................................................................13
2 材料選擇 ................................................................................................15
2.1 SU-8光阻劑 ...............................................................................15
2.2 聚二甲基矽氧烷（PDMS） .......................................................15
3 微影製程 ................................................................................................16
3.1 光罩設計與製作 .........................................................................17
3.2 晶圓片清潔（wafer cleaning） ..................................................19
3.3 光阻塗佈（spin coating） ..........................................................19
3.4 軟烤（soft bake） .......................................................................21
3.5 曝光（exposure） .......................................................................22
3.6 曝後烤（post exposure bake） ...................................................22
3.7 顯影（develop） ........................................................................23
3.8 硬烤（hard bake）......................................................................24
4 以PDMS製作微流體晶片 ....................................................................24
4.1 翻模過程 .....................................................................................25
4.2 脫模與連接器（Connector）製作..............................................26
4.3 氧電漿接合 .................................................................................26
5 微流體晶片之測詴方法與測詴結果 .....................................................28
5.1 藥品配製 .....................................................................................28
5.2 膠原蛋白濃度梯度之測詴方法 ..................................................30
5.3 濃度梯度之測詴結果 .................................................................31
5.4 FITC濃度梯度測詴 ....................................................................33
第三章 理論模型與數値方法 ................................................................................48
1基本假設與物理系統 .............................................................................48
2統御方程式 ............................................................................................49
3邊界條件 ................................................................................................49
4參數設置 ................................................................................................51
4.1流體密度 ......................................................................................52
4.2流體黏滯係數 ..............................................................................52
4.3膠原蛋白之擴散係數 ..................................................................53
5數值方法 ................................................................................................54
5.1 COMSOL Multiphysics 簡介 .....................................................54
5.2網格設置 ......................................................................................55
6數值計算之結果 .....................................................................................55
6.1速度分佈之模擬分析 ..................................................................56
6.2濃度分佈之模擬分析 ..................................................................56
6.3 FITC擴散係數之擬合分析 ........................................................58
第四章 實驗方法 .....................................................................................................74
1藥劑配製 ................................................................................................74
2細胞培養 ................................................................................................76
3實驗操作步驟 .........................................................................................79
3.1實驗前系統的準備 ......................................................................79
3.2細胞種植 ......................................................................................81
3.3以藥物建立濃度梯度與影像觀察 ...............................................82
第五章 實驗結果與討論 .........................................................................................88
1細胞靜態培養 .........................................................................................88
2 PDMCS對第一型膠原蛋白濃度梯度之反應 ........................................89
3實驗結果討論 .........................................................................................90
第六章 結論與未來展望 ....................................................................................... 100
1結論 ...................................................................................................... 100
2未來展望 .............................................................................................. 101
參考文獻 ................................................................................................................ 102

                                

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