近年來，因應各類新藥被大量開發，如何進行快速的藥物測試已然成為一個研究趨勢。人們希望可以開發出體外的藥物測試的平台，用以做為動物實驗前的先遣測試，甚至取而代之。在各針對不同疾病的測試平台中，肝組織模型的建立蔚為重要。本論文對纖維化之肝組織模型進行研究，期許可為肝纖維化之藥物開發做出貢獻。首先，由於越來越多文獻指出立體組織模型較之於傳統的平面細胞培養更能模擬真實的組織特性，故於此研究中我們採用聚甘油癸二酸脂丙烯酸脂(PGSA)支架以輔助細胞型成立體的組織結構。得益於聚甘油癸二酸脂丙烯酸脂所具有的光聚合的特性，我們得以運用3D列印製造精細的支架結構。再者，為了模擬纖維化肝組織之特性，我們共培養了肝星狀細胞(HSC)及肝細胞(HepG2)。其中，肝星狀細胞乃形成纖維化組織之關鍵。為了同時培養兩種細胞於同一系統中，我們以1:1比例混合了兩種細胞各自的培養基以做為共培養的培養基。於本文中，我們一共設計了兩種支架，分別為高通透性六層螺旋梯支架及沙漏狀支架。經過測試，兩種支架皆能輔助細胞形成穩定且可長期培養的立體組織系統。而在沙漏狀支架上，我們採用了分段式細胞接種。先遣細胞為肝星狀細胞，在接種二至三周後，支架上會形成鳥巢狀的細胞組織結構。此時，肝細胞才被接種至其中。在鳥巢狀的細胞組織結構的輔助之下，肝細胞的接種效率提升，且更能形成穩定的組織結構。而最重要的是，此結構模擬了肝纖維化的組織切片結構。最後，此模型還被證實了具有基礎的肝功能，包括白蛋白的分泌及尿素的製造。

Drug screening on a chip has been a hotspot in recent years. It is expected to provide reliable strategies for drug development and meet the 3R’s principle for experimental animals. Amongst the high demand for effective drug testing platforms, liver tissue models are urgently needed for the search and screening of new liver fibrosis drugs. In this work, to aid with drug search for the reversal of liver fibrosis, a model for liver fibrosis is established. Although 2D cell culture have already been applied in the development of new drugs, it has been shown recently that 3D culture systems are more effective in mimicking true in vivo environment. To build a 3D system for liver fibrosis, biodegradable PGSA scaffolds were fabricated via a DLP 3D-printer. Two designs were proposed based on liver lobule units: high diffusion staircase and hour glass structure. To mimic the fibrotic liver tissue, the co-culture of HepG2 and HSC is performed, and the mixture of DMEM and SteCM is chosen at a 1:1 mixing ratio for culture medium. Through sequential cell seeing, it is found that the high perfusion staircase hepatic scaffolds were not facilitating the formation of tissue with fibrotic encapsulation, while hourglass-like scaffolds exhibits such tendency. Through these models, a cluster of cells formed a section of pathological liver fibrosis encapsulated by collagen fibers. Finally, the model is tested to have the basic liver functions, including albumin production and urea secretion. Looking forward, the model is also ideal for hepatotoxicity tests and even become the base of whole organ regeneration.

摘要 1
Abstract 2
致謝 3
List of Figures 7
List of Table 9
Chapter 1. Introduction 10
1.1 Motivation 10
1.2 Introduction to Liver and Liver Diseases 10
1.2.1 Liver 12
1.2.2 Liver Cancer 14
1.2.3 Liver Fibrosis 15
1.3 Introduction to Liver Disease Models 15
1.3.1 Rodent Models 16
1.3.2 2D Tissue Culture Models 17
1.3.3 3D Tissue Culture Models 17
1.4 Introduction to Co-Cultures of Hepatic Cells 19
1.4.1 Introduction to Hepatocytes 20
1.4.2 Introduction to Hepatic Stellate Cells 21
1.4.3 Co-culture of Hepatocytes and Hepatic Stellate Cells 22
1.5 Introduction to Polymer Micro-Structure Fabrication 23
1.5.1 Reverse Molding 23
1.5.2 Additive Manufacturing 26
1.6 Introduction to Photocrosslinkable and Biocompatible Polymeric Material 28
1.6.1 Introduction to Poly(Glycerol Sebacate) (PGS) 28
1.6.2 Introduction to Poly(Glycerol Sebacate) Acrylate (PGSA) 28
Chapter 2. Experimental Methods 30
2.1 Materials and Equipments 30
2.2 Material Synthesis 31
2.2.1 Synthesis of PGS 31
2.2.2 Synthesis of PGSA 32
2.3 Design and Fabrication of Scaffolds 32
2.4 Cell Culture 33
2.5 Resazurin Reduction Assay 35
2.6 Scanning Electron Microscopy (SEM) 36
2.7 Histology 36
2.8 Functional Assessment 36
Chapter 3. Result and Discussion 37
3.1 Long Term Cultured In Vitro Liver Model 37
3.1.1 2D Co-Culture of HepG2 and HSC 37
3.1.1.1 Medium Selection 37
3.1.1.2 Co-Culture with Different Cell Ratios 39
3.1.2 3D Co-Culture of HepG2 and HSC 41
3.1.2.1 High Perfusion Hepatic Scaffold 41
3.1.2.2 Co-Culture with Different Cell Ratios 43
3.1.2.3 High Cell Seeding Density 45
3.2 Sequential Cell Seeding for Liver Fibrosis Model 46
3.2.1 Sequential Cell Seeding on High Perfusion Hepatic Scaffold 47
3.2.2 Hourglass-Like Hepatic Scaffold 49
3.2.3 Pre-Seeding of HSC 52
3.2.4 Establishment and Analysis of Liver Fibrosis Model 54
3.2.4.1 Histology 55
3.2.4.2 Liver Function Assays 57
Chapter 4. Conclusion 60
Chapter 5. Future Work 63
Chapter 6. Reference 64

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