近年來體外腸道模型發展迅速，包括可穿透性培養小室(Transwell)、腸絨毛模擬結構與微流體模型。其中微流體模型可有效模仿生物腸道內的流體與氣體環境，是公認的最先進腸道模型；然而微流體模型可提供之細胞生長貼附面積有限，不利於後續需大量細胞進行的基因體或是蛋白質體研究；此外，微流體腸道模型屬於封閉式系統，難以搭配市售設備進行上皮細胞層transepithelial electrical resistance (TEER)的量測，需於微流體模型內額外設計量測系統，因而提高模型複雜度與製造成本，不利於其普及化。有鑑於此，如何能夠藉由簡化技術製作可有效模擬腸道組織的體外腸道模型仍是一項重要挑戰。本研究應用三維列印(3D printing)技術設計並製作體外三維腸道模型 (In Vitro 3D Intestinal Model)，結合生物相容性Gel-cys/ADA水凝膠，建構可供多種腸道細胞生長之三維培養環境。此模型可搭配市售細胞培養盤與TEER量測儀器，符合一般生醫研究人員之簡便使用需求。此腸道模型所使用之Gel-cys/ADA水凝膠系統具有生物可降解特性; 相較於傳統二維細胞培養，此水凝膠可提供細胞良好三維環境進行生長與分化，並提高其分泌細胞外間質。
本研究首先探討以三維列印製備所體外腸道模型之密閉性，透過使用UV-Vis光譜分析染料水洩漏情形，證實所列印之樹酯模型經組裝後具有良好之密合度。此外我們使用alamarBlue® assay證實所列印之樹酯模型材料對於腸道細胞(Caco-2、HT29-MTX和CCD18co)不具明顯細胞毒性；透過Live/Dead staining進一步確認細胞存活度可達90%以上。接著利用alamarBlue® assay探討上述腸道細胞種植於水凝膠後的細胞活性，結果顯示所有細胞組別其培養七天後皆高於培養後一天之細胞活性1.5倍以上；Live/Dead staining結果顯示所有細胞組別於培養後七天細胞存活度皆能保持90%以上。最後我們將此列印模型結合Gel-cys/ADA水凝膠進行腸道細胞共培養探討；首先我們使用Hoechst 33342細胞核染色探討Caco-2種植於模型之分布情形，結果在Day 1觀察Caco-2分布相當均勻且細胞覆蓋率達85%。在確認細胞成功貼附後，接著量測Caco-2細胞單層之TEER值並以免疫螢光染色探討其緊密連結蛋白ZO-1表達與分佈; 經過16天時間的培養，Caco-2細胞單層之TEER值可穩定維持於150 Ohm.cm2左右，免疫螢光染色結果顯示Caco-2細胞間佈滿緊密連結蛋白ZO-1，上述結果證實培養於體外三維腸道模型之Caco-2已形成完整之細胞單層結構。最後，我們使用Alcian blue染色與Mucin 2免疫螢光染色探討模型中Caco-2、HT29-MTX與CCD18co共培養後黏蛋白Mucin生成的情形，結果觀察Mucin能夠被廣泛的生成。綜合以上結果，本研究已成功應用三維列印技術，結合生物相容性Gel-cys/ADA水凝膠，建構了一種可供多種腸道細胞生長與細胞上皮層形成之體外三維腸道模型，未來可望進一步探討此培養系統內腸道細胞間之作用，以及應用此模型探討腸道上皮層與腸道微生物之互動。

Recently, in vitro intestinal models have developed rapidly, including traditional transwell culture, intestinal villi-shaped structure models and microfluidic models. Among them, the microfluidic models can effectively mimic the fluid and gas environment of the intestine, which are recognized as the most advanced intestinal model. However, the microfluidic models limited cell growth and attachment area, which is not conducive to the subsequent need for a large number of cells for whole-genome research or proteomic research. In addition, the microfluidic systems are closed system, which is difficult to measure the transepithelial electrical resistance (TEER) value of the epithelial cell layer with commercial instruments. Therefore, an additional measurement system is designed in the microfluidic model, which increases the complexity and manufacturing cost of the model, which is not conducive to its popularization. In view of this, to make in vitro intestinal models that can effectively mimic intestinal tissues by simplifying technology is still an important challenge. In this study, a in vitro 3D intestinal model was designed with 3D printing technology to construct a variety of intestinal cell 3D culture environments, which can be used with commercial cell culture plates and TEER measurement instruments to meet the needs of general biomedical researchers. In addition, our intestinal model combines biodegradable Gel-cys/ADA hydrogel system. Compared with traditional two-dimensional cell culture, Gel-cys/ADA hydrogel provides three-dimensional environment to make cell grow and differentiate, and increases the production of cell-derived extracellular matrix.
First, we explored the water tightness of the in vitro intestinal model prepared by three-dimensional printing. Using UV-Vis spectroscopy to analyze the leakage of pigment water, we confirmed that the model has good tightness. In addition, the alamarBlue® assay was used to confirm that the resin models were not cytotoxic to intestinal cells (Caco-2, HT29-MTX and CCD18co). We used Live/Dead staining to identify the cell survival rate of the group with the model up to 90%. The alamarBlue® assay was used again to investigate the cell viability of the intestinal cells seeded on the hydrogel. The results indicated that the cell viability after 7 days of culture was at least 1.5 times higher than that 1 day of culture. The result of the Live/Dead staining showed the cell viability of each group was up to 90% after 7 days of culture. Finally, we in vitro 3D the intestinal model with hydrogel for intestinal cell co-cultivation. First, we used Hoechst 33342 nuclear staining to investigate the distribution of Caco-2 seeded on the model. The results that the distribution of Caco-2 was fairly uniform on Day 1 and the coverage rate was up to 85%. After confirming that the cells were successfully attached, we then measured the TEER value of Caco-2 cell monolayer and used immunofluorescence staining to investigate the expression and distribution of tight junction protein ZO-1. After 16 days of culture, the TEER value of Caco-2 cell monolayer remained stable at about 150 Ohm. cm2, and the result of immunofluorescence staining showed that Caco-2 was full of ZO-1 tight junction between each other. It can be seen that the Caco-2 cell monolayer structure of in vitro 3D intestine model is completely formed. Finally, we used Alcian blue staining and Mucin 2 immunofluorescence staining to investigate the production of Mucin in the model. The results observed that Mucin can be widely produced. In conclusion, this study that three-dimensional printing technology has been successfully applied, combined with biocompatible Gel-cys/ADA hydrogel, to construct an in vitro 3D intestinal model for the growth of a variety of intestinal cells and the formation of cell epithelial layers. It is expected to be further explore the role of intestinal cells in this culture system, and use this model to explore the interaction between the intestinal epithelium and intestinal microbes in the future.

摘要 1
Abstract 3
致謝 5
目錄 6
圖目錄 9
表目錄 12
第一章、緒論 13
1.1 前言 13
1.2 研究動機與目標 14
第二章、文獻回顧 17
2.1 腸道模型的現況 18
2.1.1單一培養系統腸道模型和腸類器官模型的設計概念 19
2.1.2 絨毛結構腸道模型的設計概念 20
2.1.3 微流道結構腸道模型設計概念 22
2.2 體外腸道模型總結 24
第三章、實驗材料與方法 27
3.1 實驗材料與繪圖軟體 27
3.1.1 實驗材料 27
3.1.2 繪圖軟體與硬體 28
3.2 水凝膠材料合成方法 28
3.2.1 Gelatin-cystamine(Gel-cys)合成方法 28
3.2.2 Gelatin-cystamine之氫硫基鑑定方法 29
3.2.2.1 Gelatin-cystamine還原方法 29
3.2.2.2 Ellman’s assay 29
3.2.3 Aldehyde alginate(ADA)合成方法 29
3.2.3.1 Aldehyde alginate之醛基鑑定方法 29
3.3 水凝膠的製作方法 30
3.3.1 水凝膠材料配置(Gel-cys溶液與ADA溶液) 30
3.3.2 圓餅型水凝膠製作方式 30
3.3.3 體外腸道模型內水凝膠製作方式 31
3.4 圓餅型水凝膠與體外細胞實驗之檢測方法 31
3.4.1 細胞培養與繼代 31
3.4.2 水凝膠環境中細胞活性之檢測方法 31
3.4.2.1 alamarBlue® assay 31
3.4.2.2 Live and dead assay 32
3.4.2.3 免疫螢光染色(細胞骨架與細胞核) 32
3.5 光交聯樹酯模型之印製方式 32
3.6 體外腸道模型體外細胞實驗之檢測方法 33
3.6.1 腸道模型水凝膠表面之腸道細胞細胞覆蓋程度 33
3.6.2 腸道模型之跨上皮細胞電阻測量方式 33
3.6.3 腸道模型之免疫細胞化學染色(Zonula occludens-1, Nucleus) 33
3.6.4 腸道模型之免疫細胞化學染色(Mucin 2, Nucleus) 34
3.6.5 腸道模型之免疫螢光染色(細胞骨架與細胞核) 34
3.6.6 腸道模型之Alcian blue染色 34
3.7 數據分析 35
第四章、實驗結果與討論 36
第一部分、體外腸道模型之設計與鑑定 36
4.1.1 水凝膠基座與中段圓柱設計與成品 37
4.1.3 通氣上蓋設計與成品 39
4.1.5 體外腸道模型密合性測試 40
4.1.5 光固化樹酯模型毒性測試 41
第二部分、水凝膠材料鑑定與水凝膠和腸道細胞生物相容性鑑定 45
4.2.1 Gel-cys材料的鑑定 46
4.2.2 ADA材料的鑑定 46
4.2.3 探討不同濃度水凝膠穩定性 47
4.2.4 觀察CCD18co在水凝膠內細胞存活情形 47
4.2.5 探討CCD18co在水凝膠內與Caco-2、HT29-MTX在水凝膠表面之細胞活性 48
4.2.6 觀察水凝膠內CCD18co與水凝膠表面之細胞展開程度 50
第三部分、水凝膠結合體外三維腸道模型與腸道細胞共培養其腸特徵鑑定 50
4.3.1 探討腸道模型內水凝膠表面之腸道細胞覆蓋程度 51
4.3.2 探討腸道模型之腸上皮細胞細胞單層完整性 51
4.3.2.1 探討單種腸道細胞種植於體外三維腸道模型與市售Transwell之TEER值 51
4.3.2.2 探討單種腸道細胞種植於體外三維腸道模型與Transwell之ZO-1與F-actin生成情形 52
4.3.2.3 探討多種腸道細胞種植於體外三維腸道模型之TEER值 53
4.3.2.4 探討多種腸道細胞種植於體外三維腸道模型之細胞活性 54
4.3.3 探討多種腸道細胞種植於體外三維腸道模型之黏液層生成情形 55
第五章、結論與未來展望 57
參考文獻 58

1. Belkaid, Y. and T.W. Hand, Role of the microbiota in immunity and inflammation. Cell, 2014. 157(1): p. 121-141.
2. Bradley, K.C., et al., Microbiota-driven tonic interferon signals in lung stromal cells protect from influenza virus infection. Cell reports, 2019. 28(1): p. 245-256. e4.
3. Islam, S.U., Clinical uses of probiotics. Medicine, 2016. 95(5).
4. Lievin, V., et al., Bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity. Gut, 2000. 47(5): p. 646-652.
5. Rollin, B.E., Animal rights as a mainstream phenomenon. Animals, 2011. 1(1): p. 102-115.
6. Vea, T., The learning of emotion in/as sociocultural practice: The case of animal rights activism. Journal of the Learning Sciences, 2020. 29(3): p. 311-346.
7. Broom, D.M., Animal welfare: concepts and measurement. Journal of animal science, 1991. 69(10): p. 4167-4175.
8. Kong, S., Y.H. Zhang, and W. Zhang, Regulation of intestinal epithelial cells properties and functions by amino acids. BioMed research international, 2018. 2018.
9. Majesky, M.W., et al., The adventitia: a dynamic interface containing resident progenitor cells. Arteriosclerosis, thrombosis, and vascular biology, 2011. 31(7): p. 1530-1539.
10. Gunawardene, A.R., B.M. Corfe, and C.A. Staton, Classification and functions of enteroendocrine cells of the lower gastrointestinal tract. International journal of experimental pathology, 2011. 92(4): p. 219-231.
11. Watnick, P.I. and B.-E. Jugder, Microbial control of intestinal homeostasis via enteroendocrine cell innate immune signaling. Trends in microbiology, 2020. 28(2): p. 141-149.
12. Kim, Y.S. and S.B. Ho, Intestinal goblet cells and mucins in health and disease: recent insights and progress. Current gastroenterology reports, 2010. 12(5): p. 319-330.
13. Kasendra, M., et al., Duodenum Intestine-Chip for preclinical drug assessment in a human relevant model. Elife, 2020. 9: p. e50135.
14. Kim, W. and G.H. Kim, An intestinal model with a finger-like villus structure fabricated using a bioprinting process and collagen/SIS-based cell-laden bioink. Theranostics, 2020. 10(6): p. 2495.
15. Costello, C.M., et al., Synthetic small intestinal scaffolds for improved studies of intestinal differentiation. Biotechnology and bioengineering, 2014. 111(6): p. 1222-1232.
16. Pan, F., et al., Optimization of Caco-2 and HT29 co-culture in vitro cell models for permeability studies. International journal of food sciences and nutrition, 2015. 66(6): p. 680-685.
17. Peng, H., et al., Ex vivo culture of primary intestinal stem cells in collagen gels and foams. ACS Biomaterials Science & Engineering, 2015. 1(1): p. 37-42.
18. Baulies, A., N. Angelis, and V.S. Li, Hallmarks of intestinal stem cells. Development, 2020. 147(15).
19. Gehart, H. and H. Clevers, Tales from the crypt: new insights into intestinal stem cells. Nature Reviews Gastroenterology & Hepatology, 2019. 16(1): p. 19-34.
20. Spit, M., B.-K. Koo, and M.M. Maurice, Tales from the crypt: intestinal niche signals in tissue renewal, plasticity and cancer. Open biology, 2018. 8(9): p. 180120.
21. B Sánchez, A., et al., Validation of an ex vivo permeation method for the intestinal permeability of different BCS drugs and its correlation with caco-2 in vitro experiments. Pharmaceutics, 2019. 11(12): p. 638.
22. Andersson, A.-S., et al., Influence of systematically varied nanoscale topography on the morphology of epithelial cells. IEEE transactions on nanobioscience, 2003. 2(2): p. 49-57.
23. Kim, W. and G.H. Kim, An innovative cell-printed microscale collagen model for mimicking intestinal villus epithelium. Chemical Engineering Journal, 2018. 334: p. 2308-2318.
24. Wang, Y., et al., A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium. Biomaterials, 2017. 128: p. 44-55.
25. Shim, K.-Y., et al., Microfluidic gut-on-a-chip with three-dimensional villi structure. Biomedical microdevices, 2017. 19(2): p. 37.
26. Rowland, I., et al., Gut microbiota functions: metabolism of nutrients and other food components. European journal of nutrition, 2018. 57(1): p. 1-24.
27. De Weirdt, R. and T. Van de Wiele, Micromanagement in the gut: microenvironmental factors govern colon mucosal biofilm structure and functionality. npj Biofilms and Microbiomes, 2015. 1(1): p. 1-6.
28. Albenberg, L., et al., Correlation between intraluminal oxygen gradient and radial partitioning of intestinal microbiota. Gastroenterology, 2014. 147(5): p. 1055-1063. e8.
29. Jalili-Firoozinezhad, S., et al., A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip. Nature biomedical engineering, 2019. 3(7): p. 520-531.
30. Kumar, K.K., et al., Caco-2 cell lines in drug discovery-an updated perspective. Journal of basic and clinical pharmacy, 2010. 1(2): p. 63.
31. Ferraretto, A., et al., Morphofunctional properties of a differentiated Caco2/HT-29 co-culture as an in vitro model of human intestinal epithelium. Bioscience reports, 2018. 38(2).
32. Gjorevski, N., et al., Designer matrices for intestinal stem cell and organoid culture. Nature, 2016. 539(7630): p. 560-564.
33. Dedhia, P.H., et al., Organoid models of human gastrointestinal development and disease. Gastroenterology, 2016. 150(5): p. 1098-1112.
34. Creff, J., et al., Fabrication of 3D scaffolds reproducing intestinal epithelium topography by high-resolution 3D stereolithography. Biomaterials, 2019. 221: p. 119404.
35. Castaño, A.G., et al., Dynamic photopolymerization produces complex microstructures on hydrogels in a moldless approach to generate a 3D intestinal tissue model. Biofabrication, 2019. 11(2): p. 025007.
36. Bein, A., et al., Microfluidic organ-on-a-chip models of human intestine. Cellular and molecular gastroenterology and hepatology, 2018. 5(4): p. 659-668.
37. Shah, P., et al., A microfluidics-based in vitro model of the gastrointestinal human–microbe interface. Nature communications, 2016. 7(1): p. 1-15.
38. Ulluwishewa, D., et al., Live F aecalibacterium prausnitzii in an apical anaerobic model of the intestinal epithelial barrier. Cellular microbiology, 2015. 17(2): p. 226-240.
39. Delgado-Diaz, D.J., et al., Distinct immune responses elicited from cervicovaginal epithelial cells by lactic acid and short chain fatty acids associated with optimal and non-optimal vaginal microbiota. Frontiers in cellular and infection microbiology, 2020. 9: p. 446.
40. Noel, G., et al., A primary human macrophage-enteroid co-culture model to investigate mucosal gut physiology and host-pathogen interactions. Scientific reports, 2017. 7(1): p. 1-14.
41. Shin, Y.C., et al., Three-Dimensional Regeneration of Patient-Derived Intestinal Organoid Epithelium in a Physiodynamic Mucosal Interface-on-a-Chip. Micromachines, 2020. 11(7): p. 663.
42. Sasaki, N., et al., Development of a Scalable Coculture System for Gut Anaerobes and Human Colon Epithelium. Gastroenterology, 2020. 159(1): p. 388-390. e5.
43. Sato, T., et al., Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature, 2009. 459(7244): p. 262-265.
44. Ootani, A., et al., Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nature medicine, 2009. 15(6): p. 701.
45. Lee, S.H. and J.H. Sung, Organ‐on‐a‐chip technology for reproducing multiorgan physiology. Advanced healthcare materials, 2018. 7(2): p. 1700419.
46. Kim, H.J., et al., Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow. Lab on a Chip, 2012. 12(12): p. 2165-2174.
47. Kim, H.J., et al., Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip. Proceedings of the National Academy of Sciences, 2016. 113(1): p. E7-E15.
48. Salem, D.M., M.A. Sallam, and T.N. Youssef, Synthesis of compounds having antimicrobial activity from alginate. Bioorganic chemistry, 2019. 87: p. 103-111.
49. Sarker, B., et al., Fabrication of alginate–gelatin crosslinked hydrogel microcapsules and evaluation of the microstructure and physico-chemical properties. Journal of Materials Chemistry B, 2014. 2(11): p. 1470-1482.
50. Wang, L., et al., Preparation and catalytic performance of alginate-based Schiff Base. Carbohydrate polymers, 2019. 208: p. 42-49.
51. Srinivasan, B., et al., TEER measurement techniques for in vitro barrier model systems. Journal of laboratory automation, 2015. 20(2): p. 107-126.