從大鼠心臟將細胞溶下做培養的心肌細胞為一個重要且穩定的細胞初代培養來源，用來模擬生物體心肌細胞的功能比一般細胞株更加適合與準確，而在生醫工程上，許多科學家也致力於心肌的研究與器官模型之建立，利用組織工程的概念，將人體器官用微流體整合在一個微小的晶片上甚至在未來可以和其他器官串聯，對於藥物篩選與研發具有非常重要的里程碑。
在心臟的解剖與分析中，可以發現心臟是一條肌肉帶並且具有螺旋型的旋轉與排列，而如何讓在生物體外的心肌具有功能與排列是我們所好奇的，我們在具有生物相容性的PDMS上，利用黃光微影作出各種大小不同的溝槽，來限制細胞的生長行為與約束其排列性，希望透過改變細胞生長的環境來增加心肌的收縮或功能性，並且應用在三維結構上，使其能在三維球狀PDMS上自主收縮，為一種生物體幫浦，並不像其來幫浦一樣需要外接電源或動力來驅動此微型幫浦，可以應用在許多方面，例如微流體的驅動、懸臂樑的致動，作為一種微型致動器。
本實驗利用於器官組織中常見的小鼠纖維母細胞作為初步測試，探討細胞在不同溝槽尺寸下的排列行為，再將此參數應用於大鼠初代培養，並且利用氧電漿接合的方式組成三維PDMS幫浦模型，並且於PDMS膜上培養細胞，模擬微型心臟幫浦的功能，在未來將會做為一個體外心臟模型用於藥物測試系統上。

Neonatal rat heart primary culture cardiomyocytes have been a significant model of chemical or biochemical system. Many scientists are devoted to the study of organ model and integration of different human organ model on one chip. With the concept of tissue engineering, it is an important milestone for drug screening and research.
With the anatomy of human heart, we can discover human heart is the helical ventricular myocardial band structure and cell viability and alignment are still the key issues to facilitate the cell adhesion with substrate to express proper cellular functions. Here we propose a three-dimensional culture system with different size microgrooves on the substrate with photolithography to confine and guide cardiomyocytes for realizing directional 3D artificial heart pump, and the system can be employed as a miniaturized actuators without the supplement of external energy sources.
NIH3T3 is a well-established cell line of fibroblast from mice and commonly used in biological studies so we first incubate this kind of cell to test the cell alignment with different size microgrooves. Depending on those parameters screening on 2D microgrooves, we propose a three-dimensional culture system with pre-designed PDMS microgrooves to confine cardiomyocytes with oxygen plasma treatment and photolithography. The artificial heart pump system will be employed as an in-vitro model for drug testing in the future.

摘要 i
Abstract ii
致謝 iii
目錄 iv
圖目錄 viii
表目錄 xiii
第一章 緒論 1
1.1 研究背景 1
1.2 研究動機 2
1.3 研究目的 3
第二章 文獻回顧 4
2.1 組織工程 4
2.1.1 細胞外間質與環境 5
2.1.2 水膠 8
2.2 心肌(cardiomyocyte) 11
2.3 心臟螺旋(Helix) 13
2.4 細胞排列性(anisotropic) 15
2.5.1 DTMRI 系統 16
2.5.2 PDMS微結構 18
2.5.3 Anisotropic cell sheet 19
2.5.4 具應力梯度的水膠 21
2.5 刺激 23
2.6 心臟model 27
2.7.1. 人體器官晶片 27
2.7.2. Heart pump 29
第三章 實驗設計與規劃 31
3.1 三維PDMS初步設計 32
3.1.1 帶狀PDMS 32
第四章 實驗操作流程 33
4.1 細胞微環境 33
4.1.1 細胞培養基本過程 33
4.1.2 配置培養液 34
4.1.3 細胞培養 34
4.1.4 螢光染色 35
4.2 黃光微影製程 36
4.2.1 黃光製程步驟(PDMS模具製作) 36
4.2.2 SU8膜厚量測 40
4.2.3 PDMS製備與翻膜 40
4.3 初生大鼠初代培養 41
4.3.1 實驗前準備 42
4.3.2 解剖與心臟溶解 42
4.3.3 纖維母細胞去除(pre-plating) 43
4.4 老鼠幹細胞分化 44
4.4.1 實驗藥品配置(feeder free) 45
4.4.2 心肌分化與純化 46
4.4.3 幹細胞繼代於PDMS 48
4.5 2D PDMS 製作與測試 48
4.6 電壓刺激 50
4.7 3D PDMS 製作與測試 51
4.8 實驗藥品與儀器 55
4.6.1 細胞培養 55
4.6.2 黃光微影 60
第五章 結果與討論 61
5.1 細胞培養於二維PDMS 61
5.1.1. 固定溝槽間距測試 61
5.1.2. 固定溝槽寬度測試 63
5.2 心肌培養於二維PDMS 64
5.2.1 固定溝槽間距測試 64
5.2.2 固定溝槽寬度測試 66
5.3 電壓刺激 67
5.4 心臟幫浦 68
第六章 結論 69
第七章 未來工作 71
第八章 參考文獻 72

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