由於科技的進步與快速發展，醫療技術品質也隨之提高，人們對疾病的研究與探索也愈來愈全面化。在過去生物微流體晶片方面的研究，大多都是利用二維平面化的細胞培養為基礎來進行其他生物臨床實驗如藥物測試等，但是實際上人體內組織是由三維立體的結構所組成，故這些生物測試的實驗結果會與實際上人體內的情況有出入。為了創造仿生微環境，微流體的研究結合了組織工程來模仿人體內真實的三維組織結構，並開發出具有整合系統能力的微流體生物晶片，此工程與技術的結合締造出一個能夠供於生物實驗且具有三維組織結構的微系統晶片。本研究利用流體力學的層流現象與具有生物相容性的多孔隙光固化水膠材料：Gelatin Methacrylate (GelMA) 作為細胞排列與固定封裝的方法，並搭配三維微流道的設計強化層流現象的表現，最後並以此水膠材料作為細胞排列後能夠固定封裝的媒介。在此研究，我們利用層流現象來建構一個三維的細胞排列，藉由兩種細胞的排列在微流體晶片形成肝臟組織結構，預期在晶片上呈現仿生的肝臟組織。此外，我們使用HepG2肝癌細胞以及3T3纖維母細胞來進行層流排列，模仿體內之肝細胞及內皮細胞並做三維排列共培養，並且透過微流體技術模擬肝臟內之血流流向，在體外建立一個具有仿生功能的肝臟組織結構，作為未來生物臨床實驗的研究平台。本研究利用流體力學層流現象初步建構之三維肝組織結構，其三維肝臟組織結構高度約為80~93μm，經由我們的研究期待在未來的研究上能夠有前瞻性的發展。

Due to the improvement of technology and the rapid development on our society, the quality of medicine has been enhanced. The research and exploration of people's diseases are also more comprehensive. In the past, majority of the microfluidic biochip applications based on two-dimensional planar cell culture are for biologically clinical trials such as drug testing. However, the human body tissue is composed of three-dimensional structure. Thus, the past biological results might differ from actual circumstances in the human body. In order to establish bio-mimic environments, we combined the microfluidics with the tissue engineering to approach the three-dimensional tissue structure mimicking that in the human body. We developed a micro-system chip with three-dimensional structures for targeting biological experiments. In this study, laminar flow phenomena and biocompatible porous photocurable gelatin materials were used for cell patternings and encapsulations. Gelatin Methacrylate (GelMA) was an intermediary for encapsulations and was used to fortify the cell-cell interactions after cells were patterned. We used laminar flow to set up three-dimensional patterns of two types of cells to form our bio-mimic liver tissue on the three-dimensional microfluidic chips. HepG2 cells and 3T3 fibroblast were used here. Moreover, we simulated the flow of blood through the microfluidic techniques to construct in vitro biomimetic function of the liver tissue structure. In this study, the three-dimensional structure of the liver tissue is initially patterned by the laminar flow phenomenon. The three-dimensional structure of the liver tissue is approximately 80 to 93 μm in height, which can be developed prospectively through our research in the future.

Abstract 2
摘要 3
致謝 4
目錄 5
圖目錄 7
第一章 序論 11
1.1 研究背景 11
1.1.1微機電系統與生物相容性晶片 12
1.1.2 肝臟組成與功能 12
1.1.3 肝臟疾病與肝毒性 14
1.1.4 再生醫學/組織工程 16
1.2 文獻回顧 18
1.2.1 組織培養繁殖技術 18
1.2.2 微流體系統層流特性 22
1.3 研究動機與目標 26
第二章 晶片設計與研究原理 28
2.1 設計基礎與理論 28
2.1.1 肝臟構造與肝小葉 28
2.1.2 微流體流阻分析 31
2.1.3 層流現象分析 32
2.1.4 生物支架材料 GelMA 39
2.2 晶片設計概念 40
2.2.1 細胞排列光罩定義法 44
2.2.2 層流現象分析與模擬 44
第三章 晶片製程 47
3.1 晶片製作流程 47
3.1.1 微流道母模製程 47
3.1.2 微流道晶片製程 48
3.2 製程結果 49
第四章 實驗材料與設備 50
4.1 實驗材料 50
4.1.1細胞培養 50
4.1.2生物相容性水膠 GelMA 50
4.1.3 細胞螢光染劑 52
4.1.4 晶片中生物尿素之檢測(Urea Assay) 52
4.2 實驗和儀器架設 52
第五章 實驗結果與討論 54
5.1 晶片設計原理之層流測試結果 54
5.1.1 混合細胞與水膠於層流現象 54
5.1.2 層流水膠細胞結構體進行共軛焦顯微鏡拍攝 55
5.1.3 層流水膠細胞結構體推動實驗結果 56
5.2 GelMA 結構固化曝光 57
5.2.1 GelMA 濃度與曝光時間測試 58
5.2.2 經由層流排列之水膠封裝細胞螢光圖 62
5.3 組織細胞生長情形 63
5.4 晶片內生物尿素檢測(Urea Assay) 64
第六章 結論 68
參考文獻 70

[1] V. Srinivasan, V. K. Pamula, and R. B. Fair, "An integrated digital microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids." Lab Chip, vol. 4, pp. 310-5, Aug 2004.
[2] J. Whitesides, M. Hall, R. Anchan, and A. S. LaMantia, "Retinoid signaling distinguishes a subpopulation of olfactory receptor neurons in the developing and adult mouse." J Comp Neurol, vol. 394, pp. 445-61, May 18 1998.
[3] E. M. Materne, A. G. Tonevitsky, and U. Marx, "Chip-based liver equivalents for toxicity testing--organotypicalness versus cost-efficient high throughput." Lab Chip, vol. 13, pp. 3481-95, Sep 21 2013.
[4] K. T. Patton, G. A. Thibodeau, "Anatomy and physiology." ed 7, St Louis, 2010.
[5] V. J. Navarro and J. R. Senior, "Drug-related hepatotoxicity." N Engl J Med, vol. 354, pp. 731-9, Feb 16 2006.
[6] R. S. O'Shea, S. Dasarathy, A. J. McCullough, D. Practice Guideline Committee of the American Association for the Study of Liver, and G. Practice Parameters Committee of the American College of, "Alcoholic liver disease." Hepatology, vol. 51, pp. 307-28, Jan 2010.
[7] M. Pirmohamed, A. M. Breckenridge, N. R. Kitteringham, and B. K. Park, "Adverse drug reactions." BMJ, vol. 316, pp. 1295-8, Apr 25 1998.
[8] S. Chitturi and G. C. Farrell, "Drug-Induced Liver Disease." Curr Treat Options Gastroenterol, vol. 3, pp. 457-462, Dec 2000.
[9] B. Morgan, A. L. Thomas, J. Drevs, J. Hennig, M. Buchert, A. Jivan, M. A. Horsfield, K. Mross, H. A. Ball, L. Lee, W. Mietlowski, S. Fuxuis, C. Unger, K. O'Byrne, A. Henry, G. R. Cherryman, D. Laurent, M. Dugan, D. Marmé, and W. P. Steward, "Dynamic contrast-enhanced magnetic resonance imaging as a biomarker for the pharmacological response of PTK787/ZK 222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases, in patients with advanced colorectal cancer and liver metastases: results from two phase I studies." J Clin Oncol, vol. 21, pp. 3955-64, Nov 1 2003.
[10] P. M. van Midwoud, M. T. Merema, E. Verpoorte, and G. M. Groothuis, "A microfluidic approach for in vitro assessment of interorgan interactions in drug metabolism using intestinal and liver slices." Lab Chip, vol. 10, pp. 2778-86, Oct 21 2010.
[11] P. R. Ortiz de Montellano and J. J. De Voss, "Substrate Oxidation by Cytochrome P450 Enzymes." pp. 183-245, 2005.
[12] R. Williams, S. W. Schalm, and J. G. O'Grady, "Acute liver failure: redefining the syndromes." The Lancet, vol. 342, pp. 273-275, 1993.
[13] S. Yang, K. F. Leong, Z. Du, and C. K. Chua, "The design of scaffolds for use in tissue engineering. Part I. Traditional factors," Tissue Eng, vol. 7, pp. 679-89, Dec 2001.
[14] L. G. Griffith and M. A. Swartz, "Capturing complex 3D tissue physiology in vitro." Nat Rev Mol Cell Biol, vol. 7, pp. 211-24, Mar 2006.
[15] J. L. Drury and D. J. Mooney, "Hydrogels for tissue engineering: scaffold design variables and applications." Biomaterials, vol. 24, pp. 4337-4351, 2003.
[16] K. M. Yamada and E. Cukierman, "Modeling tissue morphogenesis and cancer in 3D." Cell, vol. 130, pp. 601-10, Aug 24 2007.
[17] B. M. Baker and C. S. Chen, "Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues." J Cell Sci, vol. 125, pp. 3015-24, Jul 1 2012.
[18] S. Nemir, H. N. Hayenga, and J. L. West, "PEGDA hydrogels with patterned elasticity: Novel tools for the study of cell response to substrate rigidity." Biotechnol Bioeng, vol.
[19] F. Shen, Y. L. Cui, L. F. Yang, K. D. Yao, X. H. Dong, W. Y. Jia, and H. D. Shi, "A study on the fabrication of porous chitosan/gelatin network scaffold for tissue engineering." Polymer International, vol. 49, pp. 1596-1599, 2000.
[20] D. Huh, G. A. Hamilton, and D. E. Ingber, "From 3D cell culture to organs-on-chips." Trends Cell Biol, vol. 21, pp. 745-54, Dec 2011.
[21] Y. C. Tung, A. Y. Hsiao, S. G. Allen, Y. S. Torisawa, M. Ho, and S. Takayama, "High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array." Analyst, vol. 136, pp. 473-8, Feb 7 2011.
[22] C. T. Ho, R. Z. Lin, W. Y. Chang, H. Y. Chang, and C. H. Liu, "Rapid heterogeneous liver-cell on-chip patterning via the enhanced field-induced dielectrophoresis trap." Lab on a Chip, 6(6), pp. 724-734, 2006.
[23] D. T. Chiu, N. L. Jeon, S. Huang, R. S. Kane, C. J. Wargo, I. S. Choi, D. E. Ingber, and G. M. Whitesides, "Patterned deposition of cells and proteins onto surfaces by using three-dimensional microfluidic systems." Proc Natl Acad Sci U S A, vol. 97, pp. 2408-13, Mar 14 2000.
[24] V. L. Tsang, A. A. Chen, L. M. Cho, K. D. Jadin, R. L. Sah, S. DeLong, J. L. West, and S. N. Bhatia, "Fabrication of 3D hepatic tissues by additive photopatterning of cellular hydrogels." Faseb Journal, 21(3): p. 790-801, 2007.
[25] G. H. Underhill, A. A. Chen, D. R. Albrecht, and S. N. Bhatia, "Assessment of hepatocellular function within PEG hydrogels." Biomaterials, 28(2): p. 256-270, 2007.
[26] V. Chan, P. Zorlutuna, J. H. Jeong, H. Kong, R. Bashir, "Three-dimensional photopatterning of hydrogels using stereolithography for long-term cell encapsulation." Lab on a Chip, 10(16): p. 2062-2070, 2010.
[27] J. Y. Park, C. M. Hwang, S. H. Lee, and S. H. Lee, "Gradient generation by an osmotic pump and the behavior of human mesenchymal stem cells under the fetal bovine serum concentration gradient." Lab Chip, vol. 7, pp. 1673-80, Dec 2007.
[28] K. Gupta, D. H. Kim, D. Ellison, C. Smith, A. Kundu, J. Tuan, K. Y. Suh, and A. Levchenko, "Lab-on-a-chip devices as an emerging platform for stem cell biology." Lab on a Chip, vol. 10, pp. 2019-2031, 2010.
[29] B. G. Chung, L. A. Flanagan, S. W. Rhee, P. H. Schwartz, A. P. Lee, E. S. Monuki, and N. L. Jeon, "Human neural stem cell growth and differentiation in a gradient-generating icrofluidic device." Lab Chip, vol. 5, pp. 401-6, Apr 2005.
[30] S. Boyden, "The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes." J Exp Med, vol. 115, pp. 453-66, Mar 1 1962.
[31] D. Zicha, G. A. Dunn, and A. F. Brown, "A new direct-viewing chemotaxis chamber." J Cell Sci, vol. 99 ( Pt 4), pp. 769-75, Aug 1991.
[32] S. H. Zigmond and J. G. Hirsch, "Leukocyte locomotion and chemotaxis. New methods for evaluation, and demonstration of a cell-derived chemotactic factor." J Exp Med, vol. 137, pp. 387-410, Feb 1 1973.
[33] F. Q. Nie, M. Yamada, J. Kobayashi, M. Yamato, A. Kikuchi, and T. Okano, "On-chip cell migration assay using microfluidic channels." Biomaterials 28(27):4017–4022, 2007.
[34] K. W. Bong, K. T. Bong, D. C. Pregibon, and P. S. Doyle, "Hydrodynamic Focusing Lithography." 49(1): 87-90, 2010.
[35] S. Tasoglu, C.H. Yu, H.I. Gungordu, S. Guven, T. Vural, and U. Demirci, "Guided and Magnetic Self-Assembly of Tunable Magnetoceptive Gels." Nature Communications 5(4702), 2014.
[36] D. H. Adams, and B. Eksteen, "Aberrant homing of mucosal T cells and extra-intestinal manifestations of inflammatory bowel disease." Nature Reviews Immunology 6, 244-251, Mar 2006.
[37] A. I. Van den Bulcke, B. Bogdanov, N. De Rooze, E. H. Schacht, M. Cornelissen, and H. Berghmans, "Structural and rheological properties of methacrylamide modified gelatin hydrogels." Biomacromolecules, vol. 1, pp. 31-38, 2000.
[38] J. W. Nichol, S. T. Koshy, H. Bae, C. M. Hwang, S. Yamanlar, and A. Khademhosseini, "Cell-laden microengineered gelatin methacrylate hydrogels." Biomaterials, vol. 31, pp. 5536-5544, Jul 2010.
[39] S. C. Ramaiahgari, M. W. den Braver, B. Herpers, V. Terpstra, J. N. Commandeur, B. van de Water, and L. S. Price, "A 3D in vitro model of differentiated HepG2 cell spheroids with improved liver-like properties for repeated dose high-throughput toxicity studies." Arch Toxicol, vol. 88, pp. 1083-95, May 2014.
[40] C. T. Ho, R. Z. Lin, R. J. Chen, C. K. Chin, S. E. Gong, H. Y. Chang, H. L. Peng, L. Hsu, T. R. Yew, S. F. Chang, and C. H. Liu, "Liver-cell patterning lab chip: mimicking the morphology of liver lobule tissue." Lab Chip, vol. 13, pp. 3578-87, Sep 21 2013.
[41] H. Tekin, J. G. Sanchez, C. Landeros, K. Dubbin, R. Langer, and A. Khademhosseini, "Controlling spatial organization of multiple cell types in defined 3D geometries." Adv Mater, vol. 24, pp. 5543-7, 5542, Nov 2 2012.
[42] B. D. Plouffe, M. Radisic, and S. K. Murthy, "Microfluidic depletion of endothelial cells, smooth muscle cells, and fibroblasts from heterogeneous suspensions." Lab Chip, vol. 8, pp. 462-72, Mar 2008.