隨著科技的高度發展，佐以醫療技術的進步與發展，人們開始積極發明尋找解決所有疾病的方法。而過去生物微流體晶片方面的研究，大多是利用平面二維的細胞培養作為基準進行藥物測試，但是實際上人體內的組織結構自然是三維的立體結構，這些藥物測試的結果與實際上人體內的情況相去甚遠，為了創造更仿生的環境，研究結合了組織工程在生物上更接近人體真實的三維組織結構和微流體的全方位合一系統晶片，此兩種概念與技術的結合創造一個具有三維組織藥物檢測的微系統晶片。
本研究利用具有的生物相容性的多孔隙光固化水膠材料：gelatin methacrylate (GelMa)作為細胞的支架，並附以細胞外間質之優點加強細胞和細胞之間的交互作用，最後並以此材料作為作為細胞排列的媒介。我們建構一個三維的兩種細胞排列，三種細胞共同交互培養之肝臟組織結構並結合微流體優勢之實驗晶片，預期在晶片上模擬肝臟代謝物質的過程。我們使用肝癌細胞HepG2及纖維母細胞NIH/3T3細胞代表體內之肝細胞及內皮細胞並做三維陣列排列再加以臍帶靜脈內皮細胞HUVEC共培養，透過微流體技術模擬肝臟內之血流流向，在體外重建一個研究具有肝臟組織並作為未來新藥物測試的良好平台。本篇論文建構之三維肝組織結構，其為119 μm之高度三維肝組織結構，擁有高度的99.8 %細胞存活率；並在白蛋白的酵素免疫分析法的測試後，可證實組織結構在後續培養後，白蛋白分泌量在三維結構下與二維結構相比有高達2.44倍之成長量，表現肝功能有成長趨勢，故證實我們研究之發展前瞻性。
關鍵字：三維組織、再生醫學、生物相容性水膠、體外模型

With the advance of the science and technology, the life of human becomes more convenient but complicated too. The messy and unhealthy daily routine causes body harmful. Then people are eager to find the medical treatment for all diseases.
Most former research studies taking advantage of bio-microfluidics for drug testing focus on two-dimensional (2D) cell cultures. In reality, tissue and organs are three-dimensional (3D) structure in our bodies. To approach the biomimetic situation in vitro, this study takes advantage of microfluidic techniques and tissue engineering to build a drug-testing microsystem. Biocompatible porous photo-initiated cross-linked hydrogel, gelatin methacrylate (GelMA), was used as a scaffold material for the extracellular matrix (ECM) to enhance the cell-matrix interaction and also arrange the cell pattern. We used HepG2 cells and fibroblast NIH/3T3 to establish 3D cell structure as the model of hepatocytes and endothelial cells. Through our proposed microfluidic Labchip, we mimic the blooding pathway in vitro to reconstruct the liver function for drug testing studies.
We have constructed the 3D liver tissue on chip with the height of about 119 μm and remain the high cell viability of 99.8 % for our engineered liver tissue after our 3D rebuilding process. Also the high liver function activity, 2.4 times higher compared with the traditional 2D cell co-culture process by albumin enzyme-linked immunosorbent assay (ELISA), was demonstrated.
Keyword: Three-Dimensional Tissue, Regeneration Medicine, Biocompatible Hydrogel, In Vitro Model

目錄

摘要 II
ABSTRACT III
致謝 IV
目錄 VI
圖目錄 IX
第一章 序論 1
1.1研究背景 1
1.1.1微機電技術與實驗室晶片 1
1.1.2肝臟組成與功能 2
1.1.4再生醫學/組織工程 6
1.2文獻回顧 8
1.2.1組織培養 8
1.2.1.1電場排列法 8
1.2.1.2微流道定義法 9
1.2.1.3 GelMA水膠包埋法 10
1.2.2微流體系統產生濃度梯度 12
1.2.2.1微流體系統產生濃度梯度在生物研究上之應用 12
1.2.2.2 Christmas tree 濃度梯度產生器 13
1.2.2.3 Y 型濃度梯度產生器 14
1.3研究動機與目標 15
第二章 晶片設計與研究原理 16
2.1設計基礎與理論 16
2.1.1生物支架材料 GelMA 16
2.1.2微流體流阻分析 17
2.1.3肝臟功能檢測 18
2.1.3.1 CYP450酵素解毒系統 18
2.1.3.2合成白蛋白 18
2.1.3.3代謝氨 18
2.2.2濃度梯度產生器分析與模擬 22
第三章 晶片製程 26
3.1晶片製作流程 26
3.1.1微流道母模製程 26
3.1.2微流道晶片製程 30
3.2製程結果 32
3.2.1晶片上視圖 32
第四章 實驗材料與設備 33
4.1實驗材料 33
4.1.1細胞培養 33
4.1.2 生物相容性水膠GelMA 34
4.1.3細胞螢光染劑 35
4.1.4細胞死活檢測 35
4.1.5白蛋白酵素檢測 36
4.2實驗和儀器架設 37
第五章 實驗結果與討論 38
5.1晶片設計原理之濃度梯度測試 38
5.2 GELMA結構固化曝光初步測試 41
5.2.1 GelMA濃度與曝光時間測試 42
5.2.2曝光後之結構完整度與光源距離之測試 43
5.2.3曝光結構面積與曝光穩定度測試 43
5.2.4 細胞密度與水膠比例曝光測試 45
5.2.5水膠之通透性檢測 46
5.2.5.1灌養水膠之通透性檢測 46
5.2.5.2染劑測試 47
5.2.6排列細胞在水膠內之螢光圖 49
5.2.7細胞死活初步測試 50
5.3仿生組織之建構實驗 50
5.3.1結構尺度與間距比例之曝光成功率測試 51
5.3.2.1 GelMA混合細胞之排列二維平面圖 54
5.3.2.2 GelMA混合細胞之排列之三維立體圖 57
5.3.2.2.1共軛聚焦顯微鏡之2D平拍圖 57
5.3.2.2.2共軛聚焦顯微鏡之3D分層平拍圖 58
5.3.2.2.3共軛聚焦顯微鏡之3D合成座標圖 64
5.4 組織細胞生長情形 67
5.4.1細胞死活率測試 67
5.4.2 細胞生長活性測試 69
5.4.2.1 HepG2單養和HepG2+3T3共養之結果比較 71
5.4.2.2在NIH/3T3之共養下，有無HUVEC共養之比較 72
5.4.2.3單養HepG2與HUVEC共養之比較 73
5.5實驗結論 76
第六章 結論 77
第七章 參考文獻 78


圖目錄

圖 1.1肝臟解剖示意圖。(A)肝臟解剖圖[4] (B)血液流向圖 4
圖 1.2 肝臟疾病與之成因[4] 5
圖 1.3二維細胞與三維細胞組織比較示意圖 7
圖 1.4利用電極抓取細胞之示意圖[23] 9
圖 1.5 HEPG2和HUVEC細胞在經過電極抓取排列後的實際圖(綠色：HEPG2細胞、紅色：HUVEC細胞) [23] 9
圖 1.6 (A)PDMS與SILICON WAFER構成之結構 10
圖 1.7 HUVEC貼附於GELMA的貼附率和存活率均優於過去的水膠PEGDA 11
圖 1.8(A)利用水膠包埋細胞培養圖螢光死活圖與(B) GELMA濃度細胞存活率趨勢[25] 11
圖 1.9 HUNG 等人所設計之以對流為基礎二維濃度梯度細胞檢測 (A)晶片示意圖(B)使用三種染劑所作之測試圖,紅色染劑從左右擴散,上方為濃度梯度產生器,左右分別注入黃色與藍色染劑[36] 13
圖 1.10 利用擴散形式產生濃度梯度之 Y 形濃度梯度產生器。(A)晶片示意圖。(B)模擬之結果圖。(C)利用濃度梯度產生器來觀察細胞遷移時,細胞骨架分布情形[37] 14
圖 2.1 GELMA合成及固化示意圖(A)官能基置換(B)曝光後化學交聯[38] 16
圖 2.2 整體晶片示意圖 21
圖 2.3光罩定義細胞排列法之兩次曝光示意圖 22
圖 2.4濃度梯度產生器模擬圖 23
圖 2.5濃度梯度產生器流場分析 24
圖 2.6濃度產生器模擬電場分析 25
圖 3.1晶圓準備 27
圖 3.2旋佈光阻 27
圖 3.3一次曝光 28
圖 3.4顯影 28
圖 3.5懸塗光阻 29
圖 3.6曝光 29
圖 3.7 顯影完畢 30
圖 3.8微流道晶片製程圖 31
圖 3.9晶片實體出入口配置圖 32
圖 4.1 (A)纖維母細胞 3T3 的培養型態(B)人類肝癌細胞HEPG2(C)人類臍帶血管內皮細HUVEC 33
圖 4.2棉花狀之凍乾後GelMA原劑.................................................................................................34
圖 4.3實驗儀器架設示意圖 37
圖 5.1濃度顯示混合器 38
圖 5.2 (A)晶片色素染劑濃度梯度測試圖(B)色層分析圖 39
圖 5.3 RGB分析圖表 40
圖 5.4 RGB關係之原始值(左)與校正值(右) 41
圖 5.5 GELMA濃度測試與曝光時間圖 42
圖 5.6光源與光罩距離 43
圖 5.7多次曝光後面積測試(N=4) 44
圖 5.8曝光穩定度之統計回歸線 44
圖 5.9(A)細胞密度105個/ML與(B)106個/ML之結構圖 45
圖 5.10 GELMA灌流比較圖 47
圖 5.11染劑測試圖，各經過時間之結果圖 48
圖 5.12 肝組織細胞排列示意圖 49
圖 5.13細胞曝光死活圖，CALCEIN AM(綠LIVE)/ EDTH-1 (紅DEAD) 50
圖 5.14結構尺寸與距離大小比例之曝光後成功率 52
圖 5.15 分辨率小之結構 53
圖 5.16 3:4比例之曝光後圖 53
圖 5.17 HEPG2細胞之三維排列 55
圖5.18 NIH/3T3細胞之三維排列 56
圖5.19 HEPG2與NIH/3T3細胞之三維排列圖，HEPG2細胞組織排列(紅)，NIH/3T3細胞組織排列(綠)為 56
圖 5.20 曝光後結構之 CONFOCAL 2D平拍圖，HEPG2(紅)/3T3(綠)此兩種細胞組織之排列圖. 58
圖 5.21 灌流8小時後之 CONFOCAL 2D平拍圖，HEPG2(紅)/3T3(綠)此兩種細胞組織之排列圖 58
圖 5.22 第一組結構Z軸分層圖，HEPG2(紅)/3T3(綠)細胞組織之排列圖 59
圖 5.23第二組結構Z軸分層圖，HEPG2(紅)/3T3(綠)此兩種細胞組織之排列圖 60
圖5.24第三組結構Z軸分層圖，HEPG2(紅)/3T3(綠)此兩種細胞組織之排列圖 61
圖 5.25第四組結構Z軸分層圖，HEPG2(紅)/3T3(綠)此兩種細胞組織之排列圖 62
圖 5.26第四組結構Z軸分層圖，HEPG2(紅)/3T3(綠)此兩種細胞組織之排列圖 63
圖 5.27第一組結構之3D合成圖，HEPG2(紅)/3T3(綠)此兩種細胞組織之排列圖 64
圖 5.28第二組結構之3D合成圖，HEPG2(紅)/3T3(綠)此兩種細胞組織之排列圖 65
圖 5.29第三組結構之3D合成圖，HEPG2(紅)/3T3(綠)此兩種細胞組織之排列圖 65
圖 5.30第四組結構之3D合成圖，HEPG2(紅)/3T3(綠)此兩種細胞組織之排列圖 66
圖 5.31第五組結構之3D合成圖，HEPG2(紅)/3T3(綠)此兩種細胞組織之排列圖 66
圖 5.32細胞分別於灌流12/24/36/48小時候後死活圖，CALCEIN AM(綠LIVE)/ EDTH-1 (紅DEAD) 68
圖5.33細胞存活之分析圖表 69
圖 5.34 5倍之細胞數量分別於不同條件下之螢光圖 71
圖 5.35 HEPG2 和HEPG2+3T3之共養白蛋白分析圖表 72
圖 5.36 HEPG2+3T3和HEPG2+3T3+HUVEC共養白蛋白分泌圖 72
圖 5.37 HUVEC與HEPG2共養後之細胞伸長之構形 74
圖 5.38 HUVEC之12/24/36小時白蛋白分泌量 74
圖 5.39 HEPG2和HEPG2+HUVEC 共養之白蛋白分泌圖 75

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