臺灣博碩士論文加值系統

在此研究中開發一種腫瘤晶片(Tumor-on-a-chip)，藉由血管新生的技術，培養出可模擬體內腫瘤之體外微組織模型，以期提供可透過功能性微血管網路向腫瘤投以抗癌藥物，進行藥物篩選的研究。為了在體外實現腫瘤組織微環境，我們分別透過對流、擴散流體效應設計出了兩種可培養三維微組織的微流體平台，通過微流體系統對微腔室控制靜水壓力，氧氣張力及養分梯度做精準的控制，並找出各種培養參數如:間質滲流流速、coating時間、培養時間、氧氣濃度等不同條件下對於微血管組織培養的影響，最終我們成功在擴散質傳效應的流場中培養出三維的微組織。由實驗中發現，在以擴散主導的質傳機制下，可以成功促進血管生成(vasculogenesis)，達到35~45%血管面積比的微血管網路，並在缺氧環境(5%O2)下成功使癌組織缺氧，促使癌細胞分泌生長因子，在生長因子(VEGF)的濃度梯度下，發生腫瘤血管新生(tumor angiogenesis)，為了達成血管功能，並使血管與外部流道連接，我們透過在微流道coating一層纖維蛋白及內皮細胞，成功連接腔室內血管。由實驗結果證實，所發展的腫瘤晶片系統在培養17天之後的血管新生長度比起培養14天多了87.2%的長度，可達到1304.98μm，而在血管分支數上，培養17天(7.3 branches/vessel)也比起14天(3 branches/vessel)達到超過兩倍的分支數量，成功形成具微血管的組織結構。
透過本研究所開發出的腫瘤晶片，我們希望能在未來建立一標準的體外腫瘤模型，可以更接近體內由原來的微血管新生至腫瘤的機制，有效重現體內微環境，並通過此微流體晶片模擬體內複雜交互作用下的奈米藥物輸送，最終達到根據不同患者的篩藥結果來設計個人化的藥物治療平台，並加速前期藥物的篩選時程以及不同癌細胞間的研究。

In this study, a Tumor-on-a-chip device is developed. The goal of this project is to stimulate vasculogenic and angiogenic processes on a dual chamber device to develop a vascularized tumor model for the study of anti-cancer drugs. To develop a tumor tissue microenvironment in vitro, we design two microfluidic platforms that can develop three-dimensional microtissues using convective and diffusive mass transports. The microfluidic system controls the hydrostatic pressure, oxygen tension, and nutrient gradients inside the dual microtissue chambers. Optimization is conducted to identify culturing parameters, including interstitial flow rate, coating time, culture time, and oxygen tension. Based on the experimental study and finite element analysis, we successfully develop microvasculature in a vasculogenic chamber and induce angiogenic sprouting into adjacent tumor chamber. It is found that using diffusion-dominated mass transport, vasculogenesis can be successfully stimulated and reach 35% to 45% of the vessel area ratio. Furthermore, incubating the device in 5% oxygen incubator, SW480 tumor cells and fibroblast coculture can secret angiogenic factors. These angiogenic factor forms a concentration gradient between the dual chambers and stimulate angiogenic sprouting from vascularized tissue in the adjacent chamber. To achieve functional blood vessels and connect them with external flow channels, a layer of fibronectin and endothelial cells are lined on the microchannels. It is demonstrated that blood vessels can form connections to the external channels. Using the developed tumor-on-a-chip device, the total length of 17-day angiogenic grew vessels can be 87.2% longer than that of vessels grew in 14 days, and the length can be as long as 1304.98 μm. Furthremore, branches of sprouted vessels of 17 days also has more than twice of that in 14-day culture.
In summary, we successfully develop a microfluidic device that can induce both vasculogenic and angiogenic processes on a chip and induce angiogenic sprouting toward the microtumor. The maturation of this technology can provide a vascularized tumor model that can mimic the in vivo tumor for drug delivery studies.

口試委員會審定書 1
致謝 i
中文摘要 ii
ABSTRACT iii
目錄 iv
圖目錄 vii
表目錄 xi
第1章 緒論 1
1.1 前言 1
1.2 研究動機 1
1.3 研究目標 2
1.4 論文架構 2
第2章 文獻回顧 3
2.1 體外微血管網路之組織工程方法 3
2.1.1 纖維母細胞與內皮細胞對於血管形成的作用 3
2.1.2 應用微流體平台於血管新生 4
2.1.3 體外可灌注微血管網絡工程 5
2.2 腫瘤微環境與腫瘤微血管 8
2.2.1 腫瘤組織微環境 8
2.2.2 腫瘤組織微血管 10
2.3 藥物靶向與篩藥平台 11
2.3.1 EPR效應與奈米藥物靶向輸送 12
2.3.2 微流體晶片之藥物篩選平台 14
第3章 設計理念與研究方法 16
3.1 設計理念 16
3.1.1 微流體設計原理 16
3.1.2 可產生對流質傳效應之三維微組織培養平台設計 21
3.1.3 可產生擴散質傳效應之三維微組織培養平台設計 25
3.2 有限元素法之微流體流場模擬分析 30
3.2.1 有限元素分析之建模 30
3.2.2 有限元素分析之統御方程式 32
3.2.3 有限元素分析之邊界條件 35
3.2.4 有限元素分析之材料屬性 36
3.2.5 有限元素分析之網格 37
第4章 平台開發與實驗方法 39
4.1 微流體平台系統之製程開發 39
4.1.1 微流體晶片製程 39
4.1.2 晶片光罩繪製與製作 40
4.1.3 黃光微影製程 41
4.1.4 微流道軟微影製程 50
4.1.5 微流道系統量測 53
4.1.6 微流體平台系統架設 54
4.2 微流體平台之流體總填充時間分析 56
4.3 細胞培養及生物技術 56
4.3.1 細胞培養技術 57
4.3.2 細胞固定與免疫螢光染色 58
4.4 血管新生量化分析 58
第5章 實驗結果與討論 60
5.1 微流體平台流場分析結果 60
5.1.1 對流質傳效應之三維微組織培養平台流場 60
5.1.2 擴散質傳效應之三維微組織培養平台流場 63
5.2 微流體平台總填充時間分析結果 65
5.2.1 對流流體效應之三維微組織培養平台總充填時間分析 65
5.2.2 擴散流體效應之三維微組織培養平台總填充時間分析 68
5.3 對流質傳效應之三維微組織培養結果 72
5.4 擴散質傳效應之三維微組織培養結果 74
5.4.1 擴散質傳效應之三維微組織—血管新生(Angiogenesis) 75
5.4.2 擴散質傳效應之三維微組織—腫瘤微血管(Tumor Angiogenesis) 86
5.4.3 擴散質傳效應之三維微組織——血管吻合術(Anastomosis) 95
第6章 結論與未來展望 100
6.1 結論 100
6.2 未來展望 100
Reference 101

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