隨著人類生活習慣的改變，不良的作息、過度精緻的飲食習慣等等更是造成身體產生病變的主因，因此人們開始積極地尋找建立體外人體組織之平台，除了能更了解細胞的生長情形，同時可對其進行藥物測試、生長激素刺激等，尋找最佳的治療藥物、觀察不同生長激素之於細胞的影響，替代人體實驗，了解體內組織運作情形。微流體晶片提供了一個適合細胞生長的環境，過去的研究多以二維平面化的細胞培養為基礎來進行生物臨床實驗，然而實際上人體的組織結構是複雜的三維立體結構，在二維細胞上進行測試的結果仍與實際人體內的情況相去甚遠，為了創造更貼近體內環境的仿生平台，研究結合了組織工程和微流體的概念，創造一個三維組織藥物檢測的微系統晶片。
本研究提出以介電泳力排列肝癌細胞與小鼠纖維母細胞，重建體內組織環境，觀察在不同濃度的生長激素下癌細胞之轉移情形，同時也可透過此為流體平台尋找癌細胞加速轉移的誘因。透過生物相容性之光固化水膠材料（gelatin methacrylate，簡稱GelMA）作為細胞支架，並藉由此材料之光固化特性，將細胞固定於以介電泳力排列後之位置，預期在晶片上呈現仿生的肝癌細胞與表皮細胞排列情形。
本實驗以胎牛血清作為癌細胞遷移誘因，血清及培養液經濃度梯度產生器可產生遞減之濃度，流入細胞培養區外的通道，經由擴散機制進入仿生細胞組織，可同時於晶片上觀察不同濃度生長激素下促使癌細胞的遷移距離和數量，作為未來觀察促使癌細胞遷移之誘因之平台，實驗結果顯示，在血清濃度為10%時，細胞於孔盤實驗上隻存活率最高，在培養48小時後，可高於90%；於本研究之晶片上進行的細胞遷移實驗，可以觀察到，在比較血清濃度為0%、5%和10%時，10%濃度下的細胞遷移距離最遠且數目最多，遷移距離約為100µm至200µm。

Irregular lifestyle and excessively refined eating habits lead to body diseases. To study these phenomena and relationships, people look for establishing platforms to either reconstruct or culture in-vitro human tissue, study the interaction between cells, and be applied to drug treatment. Microfluidic chips could provide a suitable microenvironment for cell growth. In the past, most microfluidic cell/tissue/organ chips are based on two-dimensional planar cells cultured for biologically clinical trials such as drug testing. However, human organs and tissues are composed of complex three-dimensional（3D）structure. Thus, the research studies here focus on establishing an in-vitro biomimetic microenvironment taking advantage of 3D-tissue reconstruction and microfluidic culturing and gradients.
In this master study, fibroblast cells with the pumped microfluidics were encapsulated and patterned to form a 3D structure to mimic the vascular wall and structure. The dielectrophoresis force （DEP） and photocrosslinked gelatin methacryloyl (GelMA) hydrogel were used for this 3D construction of fibroblast cells. Then, liver cancer cells were positioned and co-cultured in-between two-side 3D fibroblast cells. The invasion of liver cancer cells in individual culture chamber under different growth factor concentrations was observed and quantitatively studied. Through our liver cancer-cell invasion micro-environment LabChip, the blooding pathway with liver tumor tissue was reconstructed and mimicked in vitro to study the invasion of tumor tissue.
In our experimental results, liver cancer cells have optimal cell viability with the culture medium of 10% FBS. For on-chip culturing of 48 hours, cell viability was still maintained higher than 90%. With the comparison of different FBS concentrations varied from 0% to 10%, the largest migrating distance of liver cancer cells, about 100 µm to 200 µm, was observed for the medium of 10% FBS.

Abstract II
摘要 IV
目錄 V
圖目錄 VIII
表目錄 XIII
第一章 緒論 1
1.1研究背景 1
1.1.1實驗室晶片 2
1.1.2肝臟組織與功能 3
1.1.3肝臟疾病與肝毒性 5
1.1.4 癌細胞轉移 7
1.1.5組織工程 9
1.2研究動機與目的 11
1.3文獻回顧 12
1.3.1組織培養 12
1.3.2濃度梯度產生器 15
1.3.3粒子操控技術 17
1.3.3 細胞遷移 25
第二章 系統理論與晶片設計 26
2.1 設計基礎及理論 26
2.1.1介電泳理論 26
2.1.2擴散理論 30
2.1.3微流體晶片設計理論 32
2.1.4生物支架材料GelMA 35
2.2晶片設計概念 37
2.2.1細胞排列光罩定義法 39
2.2.2濃度梯度產生器模擬 42
2.2.3電極設計及模擬 43
第三章 微流體晶片製程 45
3.1電極基板製作流程 45
3.2晶片製作流程 47
3.2.1微流道母模製程 47
3.2.2微流道晶片製程 49
第四章 實驗材料與方法 51
4.1實驗材料 51
4.1.1細胞培養 51
4.1.2 低導電度溶液 53
4.1.3生物相容性材料水膠GelMA 53
4.1.4細胞螢光染劑 56
4.1.5細胞死活檢測 56
4.1.6 細胞存活率與細胞毒性試驗（Alamar Blue cell viability assay） 57
4.2實驗前置準備 59
4.2.1晶片清洗 59
4.2.2儀器架設 60
4.3實驗操作流程 62
第五章 實驗結果與討論 64
5.1晶片濃度梯度測試結果 64
5.2水膠GelMA曝光測試結果 67
5.3細胞存活率 69
5.3.1 Alamar Blue Assay 69
5.3.2 細胞於GelMA中之存活率 70
5.3.3細胞於不同電壓及操作時間下之存活率 71
5.4細胞排列情形 72
5.4.1晶片二維平拍圖 72
5.4.2共軛聚焦顯微鏡之分層平拍圖 74
5.4.3細胞遷移率測試 75
5.4.5細胞密度 77
第六章 結論與未來展望 78
參考文獻 80

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