在全世界為數眾多惡性腫瘤的患者中，肺癌是長年以來許多患者的噩夢之一，想當然每年也有許多患者死於肺癌。由許多臨床證據顯示出，對於癌症患者而言，癌轉移才是造成他們死亡的主要原因。為了研究並了解癌轉移的機制，重建惡性腫瘤之環境的體外裝置被視為相對簡單並可模仿癌細胞與其周圍體細胞複雜又多樣化的互動的方法之一。然而，目前為止所發展的裝置通常只在靜止狀態下做培養或只包含一至二種細胞以至於無法完整的複製癌細胞之環境且製作過程繁瑣又費時。對此，本篇研究將針對是否流動系統及立體培養會影響癌細胞以及共培養、細胞激素如何影響癌細胞行為等兩大方向作探討。我們設計以微流體作為基礎的裝置結合肺癌細胞A549和肺微血管內皮細胞以創造更完善的腫瘤環境。該裝置的主要特點為我們設計了可拋棄式的卡匣，該卡匣在裝置中主要為癌細胞培養的位置。對應上述之不同條件，我們可以任意調整卡匣中癌細胞的環境以達到隨插即用的功能和即時且可數量化的癌細胞轉移或生長的數據。此外該卡匣也與循環流動系統結合以建構體外肺癌平台。截至目前為止，該裝置已經完成了：(1)A549/GFP細胞的平面培養與細胞激素TGF-β1投放比較；(2) A549/GFP細胞與肺微血管內皮細胞之共培養；(3)A549/GFP細胞在膠原蛋白中的立體培養；(4)A549/GFP細胞在流動系統中的收集。未來，我們也計畫將纖維母細胞或巨噬細胞結合癌細胞共同培養於裝置中並加以投放抗癌藥物以創造出更完善的癌細胞環境利於未來之癌轉移研究。

Lung cancer is one of the most common malignancies and the leading causes of cancer-related death in the world. Many clinical data has shown that cancer metastasis is the major reason that causes the death of patients. To investigate the mechanism of metastasis, reconstruction of tumor microenvironment in vitro is considered as an easily accessible approach that can mimic the complex and multiple interactions among lung tumor cells and other surrounding cells. However, the existing devices usually contain no flow dynamic systems which are not able to represent the tumor microenvironment. Moreover, the manufacturing and operation processes are complex and time-consuming. Therefore, the goals of this study are to understand if a flow dynamic system and 3D culture can affect the growth conditions of the carcinoma cells and if a co-culture of multiple cells and adding of grow factors will alter the behaviors of the tumor cells. Here, we developed a microfluidic-based platform that can culture lung cancer cells and endothelial cells in the same format to mimic a tumor microenvironment. We developed a technique to fabricate a microfluidic-based platform that contains multiple disposable microfluidic clips. These clips could be adjusted to meet certain culture condition and plug-and-play after observation so that the cancer cells migration results can be in time and quantitatively obtained. In addition, we connected the tumor chip with the circulating system to form an in vitro human lung platform. So far, we demonstrated: (1) The capability of the A549/GFP cell culture in the tumor chip with TGF-β1 treatment. (2) The co-culture of A549/GFP and HPMECs cells in tumor chip. (2) The proliferation and migration of A549/GFP cells in 3D collagen scaffold. (3) The capturing and culturing of A549/GFP cells in flow dynamic system. For the future applications, fibroblasts or macrophage cells can be introduced in the system to construct more advanced tumor microenvironment for drug development research. For example, antineoplastic drugs can be administrated through the circulating system in our platform.

Table of Content
Chapter 1: Introduction & paper review---------------------------1
1-1 Review: 2D, 3D microfluidic devices--------------------------1
1-1-1 3D structure-----------------------------------------------2
1-1-2 Microchannel structure-------------------------------------3
1-1-3 Membrane application---------------------------------------3
1-2 Lack of Multicellular interaction----------------------------4
1-2-1 Fibroblasts------------------------------------------------5
1-2-2 Endothelial cells------------------------------------------6
1-3 Tumor microenvironments--------------------------------------8
1-3-1 Epithelial-mesenchymal transition (EMT)--------------------8
1-3-2 Cancer cell migrations-------------------------------------8
1-3-3 Intravasation---------------------------------------------10
1-3-4 Current invasion model------------------------------------11
Chapter 2: Approaches-------------------------------------------13
2-1 Device design-----------------------------------------------13
2-1-1 Device formation by a laser cutter------------------------17
2-2 Cell culture------------------------------------------------18
2-2-1 Sterilization---------------------------------------------18
2-3 Cytokines assay---------------------------------------------19
2-3-1 Live and dead staining------------------------------------19
2-4 Pretesting procedures---------------------------------------19
2-4-1 Flow testing----------------------------------------------19
2-4-2 HeLa cells testing----------------------------------------20
2-5 Experiment procedures---------------------------------------20
2-5-1 A549/GFP 2D cell culture----------------------------------20
2-5-2 A549/GFP 3D cell culture----------------------------------21
3-1 Pretesting results------------------------------------------22
3-1-1 Flow testing----------------------------------------------22
3-1-2 HeLa cells testing----------------------------------------23
3-2 Experiment results------------------------------------------23
3-2-1 A549 2D cell culture--------------------------------------23
3-2-2 A549/GFP 2D mono-cell culture-----------------------------27
3-2-3 A549/GFP 2D mono-cell culture with TGF-β1 in medium------29
3-2-4 A549/GFP 2D co-culture with HPMECs------------------------30
3-2-5 A549 3D cell culture on alginate surface------------------33
3-2-6 A549 3D cell culture on the collagen surface--------------34
3-2-7 A549/GFP 3D cell culture in collagen hydrogel-------------36
3-2-8 A549/GFP 3D cell culture in thin collagen-----------------39
3-2-9 A549/GFP cells collection in medium container-------------45
Chapter 4: Conclusions------------------------------------------49
Chapter 5: Future applications----------------------------------51
5-1 Advance tumor microenvironment constructions----------------51
5-2 Antineoplastic drugs administrations------------------------52
Chapter 6: Reference--------------------------------------------53

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