細胞層片培養技術已被廣泛應用於修補各種器官創傷，例如在眼睛、心臟、膀胱和腎臟等。然而，由於單層細胞過於脆弱，不易於夾取和放置於受傷區域，因此，一個強韌和易於操作的基底是非常需要的。在本篇研究中，我們使用絲質蛋白作為生物材料，因為它擁有很強的機械性質和生物相容性。而絲質蛋白可經由蠶絲脫除絲膠處理取得。本論文首先決定哪種脫膠方式和絲質蛋白濃度可以有較好的細胞生長和抗拉強度。考量到細胞生長、抗拉強度、操作方便性和成本，以3 mg/mL的絲質蛋白濃度作為後續實驗的操作濃度。其次，為了增加細胞在絲質蛋白膜上的貼附能力，我們將膠原蛋白覆蓋於膜上，結果顯示含有膠原蛋白的絲質膜可促進細胞存活率。接著，我們將這層絲質蛋白膜應用於建立空氣-液體介面之細胞培養系統。這個系統可以讓細胞頂端暴露在空氣中而養分則從細胞的底部獲得，用以模擬體內上皮層的真實結構和功能。我們比較細胞分別在浸潤的培養基和空氣-液體介面培養之下，發現緊密連接蛋白(ZO-1)和絨毛蛋白(villin)在細胞內的位置無顯著差異。最後，為了證明此種利用絲質蛋白膜建立的空氣-液體介面培養方式有其可用性，我們將其應用於觀察綠膿桿菌PAO1侵入大腸癌細胞C2BBe1的情形。透過共軛焦顯微鏡的圖顯示出清楚的細菌和細胞之相對應關係。總結上述，絲質蛋白膜不僅可以當作強韌的細胞層片基底，還可以應用於建立空氣-液體介面的細胞培養系統。

Cell sheet technology has been widely applied to treat damaged organs such as the eyes, heart, bladder, and kidney. However, cell sheets are generally fragile and are difficult to handle and attach to damaged sites. Therefore, a robust and easy-to-hold substratum is required for generating cell sheets. In this study, we utilized silk fibroin as a biomaterial owing to its mechanical properties and biocompatibility. Fibroin is a water insoluble protein which can be extracted from silk fibers by degumming processes. This study first determined the suitable degumming method and fibroin concentration to provide the better cell growth and the higher tensile strength. The results showed that 3 mg/mL fibroin derived from high temperature water degumming is the ideal concentration for the following experiments in consideration of cell growth, tensile strength, handling problem, and cost. Secondly, to improve cell adhesion on fibroin films, we coated the fibroin films with collagen. The coating process yielded higher cell viability than non-coating films. Thirdly, we applied the fibroin film to develop air-liquid interface (ALI) culture, where cells are exposed directly to the air apically with nutrients supplied basally. Cells grown in ALI culture resemble the real structures and functions in the epithelium of our bodies. The results showed that the localization of zonula occludens-1 and villin had no significant difference between ALI culture and submerged culture. Finally, to demonstrate the usability of the fibroin film-based ALI culture, we used it as a bacterial invasion model by infecting C2BBe1 cell sheet with Pseudomonas aeruginosa PAO1. The confocal images showed clear interactions between cells and bacteria. To conclude, silk fibroin films can not only serve as a strong basis for cell sheet but also be applied to develop ALI culture.

Abstract I
中文摘要 II
Acknowledgement III
Abbreviations IV
Table of contents VI
List of tables VIII
List of figures IX
List of appendixes XI
1. Introduction 1
2. Materials and methods 6
2.1 Preparation of fibroin films 6
2.1.1 Degumming of silk 6
2.1.2 Dialysis of fibroin solution 6
2.1.3 Measurement of fibroin concentration 7
2.1.4 Formation of fibroin films 7
2.2 Cell culture 8
2.2.1 Maintenance of cells 8
2.2.2 Subculture of C2BBe1 cells 8
2.3 alamarBlue assay for quantification of cell viability 8
2.3.1 Preparation of cells seeded on two different degummed fibroin films 8
2.3.2 Preparation of cells seeded on collagen-coated fibroin films 9
2.3.3 alamarBlue assay 9
2.4 Live/dead cell viability assay 10
2.5 Tensile test of fibroin films 10
2.6 Establishment of ALI culture using fibroin films 11
2.6.1 Using a floating material 11
2.6.2 Using a hollow support cylinder 11
2.6.3 Using a semi-solid material 11
2.6.4 Using a porous material 11
2.7 Immunofluorescence staining 12
2.8 RNA extraction and RT-PCR 13
2.9 Bacterial strain and culture 14
2.10 Infection of cultured C2BBe1 cells with P. aeruginosa PAO1 14
2.11 Statistical analyses 14
3. Results 15
3.1 Different transparency of fibroin films formed from using high temperature water degumming method and boiling sodium carbonate solution degumming method 15
3.2 Growth of C2BBe1 cells on films formed using different concentrations of fibroin derived from boiling sodium carbonate solution degumming and high temperature water degumming 15
3.3 Tensile test of different concentrations of fibroin films 16
3.4 Improvement of cell adhesion to fibroin films by coating with collagen 17
3.5 Comparison of four different ALI culture systems 18
3.6 Comparison of the established ALI culture with commercial transwells 19
3.7 Immunofluorescence staining of tight junction protein ZO-1 in C2BBe1 cells cultured in the submerged condition and the ALI condition 20
3.8 Immunofluorescence staining of villin in C2BBe1 cells cultured in the
submerged condition and the ALI condition 20
3.9 Comparison of mRNA expression of ZO-1 and villin in C2BBe1 cells cultured in the submerged condition and the ALI condition 21
3.10 Effects of Pseudomonas aeruginosa PAO1 strain invasion on C2BBe1 cells 21
4. Discussion 23
5. References 28

1. Nishida K, Yamato, M., Hayashida, Y., Watanabe, K., Yamamoto, K., Adachi, E., Nagai, S., Kikuchi, A., Maeda, N., Watanabe, H., Okano, T., Tano, Y. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med. 2004;351(12):1187-1196.
2. Masuda S, Shimizu, T., Yamato, M., Okano, T. Cell sheet engineering for heart tissue repair. Advanced Drug Delivery Reviews. 2008;60(2):277-285.
3. Yamato M, Okano, T. Cell sheet engineering. Materials Today. 2004;7(5):42-47.
4. Tang Z, Akiyama, Y., Okano, T. Temperature-responsive polymer modified surface for cell sheet engineering. Polymers. 2012;4(3):1478.
5. Shimizu T, Yamato, M., Kikuchi, A., Okano, T. Two-dimensional manipulation of cardiac myocyte sheets utilizing temperature-responsive culture dishes augments the pulsatile amplitude. Tissue Eng. 2001;7(2):141-151.
6. Wei Q, Reidler, D., Shen, M. Y., Huang, H. Keratinocyte cytoskeletal roles in cell sheet engineering. BMC Biotechnol. 2013;13:17.
7. Patel NG, Zhang, G. Stacked stem cell sheets enhance cell-matrix interactions. Organogenesis. 2014;10(2):170-176.
8. Haraguchi Y, Shimizu, T., Sasagawa, T., Sekine, H., Sakaguchi, K., Kikuchi, T., Sekine, W., Sekiya, S., Yamato, M., Umezu, M., Okano, T. Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro. Nat Protoc. 2012;7(5):850-858.
9. Tadakuma K, Tanaka, N., Haraguchi, Y., Higashimori, M., Kaneko, M., Shimizu, T., Yamato, M., Okano, T. A device for the rapid transfer/transplantation of living cell sheets with the absence of cell damage. Biomaterials. 2013;34(36):9018-9025.
10. Altman GH, Diaz, F., Jakuba, C., Calabro, T., Horan, R. L., Chen, J., Lu, H., Richmond, J., Kaplan, D. L. Silk-based biomaterials. Biomaterials. 2003;24(3):401-416.
11. Vepari C, Kaplan, D. L. Silk as a biomaterial. Prog Polym Sci. 2007;32(8-9):991-1007.
12. Omenetto FG, Kaplan, D. L. A new route for silk. Nat Photon. 2008;2(11):641-643.
13. Wang Y, Rudym, D. D., Walsh, A., Abrahamsen, L., Kim, H. J., Kim, H. S., Kirker-Head, C., Kaplan, D. L. In vivo degradation of three-dimensional silk fibroin scaffolds. Biomaterials. 2008;29(24-25):3415-3428.
14. Soong HK, Kenyon, K. R. Adverse reactions to virgin silk sutures in cataract surgery. Ophthalmology. 1984;91(5):479-483.
15. Zaoming W, Codina, R., Fernandez-Caldas, E., Lockey, R. F. Partial characterization of the silk allergens in mulberry silk extract. Journal of investigational allergology & clinical immunology. 1996;6(4):237-241.
16. Yamaguchi K, Kikuchi, Y., Takagi, T., Kikuchi, A., Oyama, F., Shimura, K., Mizuno, S. Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis. Journal of molecular biology. 1989;210(1):127-139.
17. Tanaka K, Kajiyama, N., Ishikura, K., Waga, S., Kikuchi, A., Ohtomo, K., Takagi, T., Mizuno, S. Determination of the site of disulfide linkage between heavy and light chains of silk fibroin produced by Bombyx mori. Biochimica et biophysica acta. 1999;1432(1):92-103.
18. Tanaka K, Inoue, S., Mizuno, S. Hydrophobic interaction of P25, containing Asn-linked oligosaccharide chains, with the H-L complex of silk fibroin produced by Bombyx mori. Insect biochemistry and molecular biology. 1999;29(3):269-276.
19. Zhou CZ, Confalonieri, F., Jacquet, M., Perasso, R., Li, Z. G., Janin, J. Silk fibroin: structural implications of a remarkable amino acid sequence. Proteins. 2001;44(2):119-122.
20. Murphy AR, Kaplan, D. L. Biomedical applications of chemically-modified silk fibroin. J Mater Chem. 2009;19(36):6443-6450.
21. Pérez-Rigueiro J, Viney, C., Llorca, J., Elices, M. Mechanical properties of single-brin silkworm silk. Journal of Applied Polymer Science. 2000;75(10):1270-1277.
22. Cunniff PM, Fossey, S. A., Auerbach, M. A., Song, J. W., Kaplan, D. L., Adams, W. W., Eby, R. K., Mahoney, D., Vezie, D. L. Mechanical and thermal properties of dragline silk from the spider Nephila clavipes. Polymers for Advanced Technologies. 1994;5(8):401-410.
23. Pins GD, Christiansen, D. L., Patel, R., Silver, F. H. Self-assembly of collagen fibers. Influence of fibrillar alignment and decorin on mechanical properties. Biophysical journal. 1997;73(4):2164-2172.
24. Engelberg I, Kohn, J. Physico-mechanical properties of degradable polymers used in medical applications: a comparative study. Biomaterials. 1991;12(3):292-304.
25. Kundu B, Rajkhowa, R., Kundu, S. C., Wang, X. Silk fibroin biomaterials for tissue regenerations. Advanced Drug Delivery Reviews. 2013;65(4):457-470.
26. Bettinger CJ, Cyr, K. M., Matsumoto, A., Langer, R., Borenstein, J. T., Kaplan, D. L. Silk Fibroin Microfluidic Devices. Advanced materials (Deerfield Beach, Fla.). 2007;19(5):2847-2850.
27. Santin M, Motta, A., Freddi, G., Cannas, M. In vitro evaluation of the inflammatory potential of the silk fibroin. J Biomed Mater Res. 1999;46(3):382-389.
28. Meinel L, Hofmann, S., Karageorgiou, V., Kirker-Head, C., McCool, J., Gronowicz, G., Zichner, L., Langer, R., Vunjak-Novakovic, G., Kaplan, D. L. The inflammatory responses to silk films in vitro and in vivo. Biomaterials. 2005;26(2):147-155.
29. Liu J, Lawrence, B. D., Liu, A., Schwab, I. R., Oliveira, L. A., Rosenblatt, M. I. Silk fibroin as a biomaterial substrate for corneal epithelial cell sheet generation. Invest Ophthalmol Vis Sci. 2012;53(7):4130-4138.
30. Krunkosky TM, Jordan, J. L., Chambers, E., Krause, D. C. Mycoplasma pneumoniae host–pathogen studies in an air–liquid culture of differentiated human airway epithelial cells. Microbial Pathogenesis. 2007;42(2):98-103.
31. Martin-Belmonte F, Mostov, K. Regulation of cell polarity during epithelial morphogenesis. Current opinion in cell biology. 2008;20(2):227-234.
32. Roignot J, Peng, X., Mostov, K. Polarity in mammalian epithelial morphogenesis. Cold Spring Harbor perspectives in biology. 2013;5(2).
33. Whitcutt MJ, Adler, K. B., Wu R. A biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells. In Vitro Cellular & Developmental Biology. 1988;24(5):420-428.
34. Pezzulo AA, Starner, T. D., Scheetz, T. E., Traver, G. L., Tilley, A. E., Harvey, B. G., Crystal, R. G., McCray, P. B., Zabner, J. The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia. American Journal of Physiology - Lung Cellular and Molecular Physiology. 2011;300(1):L25-31.
35. de Jong PM, van Sterkenburg, M. A., Hesseling, S. C., Kempenaar, J. A., Mulder, A. A., Mommaas, A. M., Dijkman, J. H., Ponec, M. Ciliogenesis in human bronchial epithelial cells cultured at the air-liquid interface. American journal of respiratory cell and molecular biology. 1994;10(3):271-277.
36. Ootani A, Toda, S., Fujimoto, K., Sugihara, H. An air-liquid interface promotes the differentiation of gastric surface mucous cells (GSM06) in culture. Biochemical and biophysical research communications. 2000;271(3):741-746.
37. Nossol C, Diesing, A., Walk, N., Faber-Zuschratter, H., Hartig, R., Post, A., Kluess, J., Rothkötter, H., Kahlert, S. Air–liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC). Histochemistry and Cell Biology. 2011;136(1):103-115.
38. Karam SM, Leblond, C. P. Dynamics of epithelial cells in the corpus of the mouse stomach. II. Outward migration of pit cells. The Anatomical record. 1993;236(2):280-296.
39. Bradbury MWB. Introduction to the Blood-Brain Barrier. Methodology, Biology and Pathology. J Anat. 1999;194(Pt 1):153-157. doi:110.1046/j.1469-7580.1999.194101531.x.
40. Kramer N, Walzl, A., Unger, C., Rosner, M., Krupitza, G., Hengstschlager, M., Dolznig, H. In vitro cell migration and invasion assays. Mutation research. 2013;752(1):10-24.
41. Mitchell G, Grondin, G., Bilodeau, G., Cantin, A. M., Malouin, F. Infection of polarized airway epithelial cells by normal and small-colony variant strains of Staphylococcus aureus is increased in cells with abnormal cystic fibrosis transmembrane conductance regulator function and is influenced by NF-kappaB. Infection and immunity. 2011;79(9):3541-3551.
42. Rajkhowa R, Gupta, V. B., Kothari, V. K. Tensile stress–strain and recovery behavior of Indian silk fibers and their structural dependence. Journal of Applied Polymer Science. 2000;77(11):2418-2429.
43. Gulrajani ML. Degumming of silk. Review of Progress in Coloration and Related Topics. 1992;22(1):79-89.
44. Panilaitis B, Altman, G. H., Chen, J., Jin, H. J., Karageorgiou, V., Kaplan, D. L. Macrophage responses to silk. Biomaterials. 2003;24(18):3079-3085.
45. Pérez-Rigueiro J, Viney, C., Llorca, J., Elices, M. Mechanical properties of silkworm silk in liquid media. Polymer. 2000;41(23):8433-8439.
46. Dhyani V, Singh, N. Controlling the cell adhesion property of silk films by graft polymerization. ACS Appl Mater Interfaces. 2014;6(7):5005-5011.
47. Hu K, Lv, Q., Cui, F. Z., Feng, Q. L., Kong, X. D., Wang, H. L., Huang L. Y., Li T. Biocompatible fibroin blended films with recombinant human-like collagen for hepatic tissue engineering. Journal of Bioactive and Compatible Polymers. 2006;21(1):23-37.
48. Coudrier E, Arpin, M., Dudouet, B., Finidori, J., Garcia, A., Huet, C., Pringault, E., Robine, S., Sahuquillo-Merino, C., Louvard, D. Villin as a structural marker to study the assembly of the brush border. Protoplasma. 1988;145(2):99-105.
49. L. GSCM. Comparative evaluation of the various methods of degumming silk. Indian Journal of Fibre & Textile Research. 1994;19:76-83.
50. Teh TK, Toh, S. L., Goh, J. C. Optimization of the silk scaffold sericin removal process for retention of silk fibroin protein structure and mechanical properties. Biomed Mater. 2010;5(3):35008.
51. Wray LS, Hu, X., Gallego, J., Georgakoudi, I., Omenetto, F. G., Schmidt, D., Kaplan, D. L. Effect of processing on silk-based biomaterials: reproducibility and biocompatibility. Journal of biomedical materials research. Part B, Applied biomaterials. 2011;99(1):89-101.
52. Watt SW, McEwen, I. J., Viney, C. Stability of molecular order in silkworm silk. Macromolecules. 1999;32(25):8671-8673.
53. Jin HJ, Park, J., Karageorgiou, V., Kim, U. J., Valluzzi, R., Cebe, P., Kaplan, D.  L. Water-stable silk films with reduced β-sheet content. Advanced Functional Materials. 2005;15(8):1241-1247.
54. Lu Q, Hu, X., Wang, X., Kluge, J. A., Lu, S., Cebe, P., Kaplan, D. L. Water-insoluble silk films with silk I structure. Acta Biomater. 2010;6(4):1380-1387.
55. Leal-Egaña A, Lang, G., Mauerer, C., Wickinghoff, J., Weber, M., Geimer, S. and Scheibel, T. Interactions of fibroblasts with different morphologies made of an engineered spider silk protein. Advanced Engineering Materials. 2012;14(3):B67-B75.
56. Leal-Egana A, Scheibel, T. Interactions of cells with silk surfaces. Journal of Materials Chemistry. 2012;22(29):14330-14336.
57. Bray LJ, Suzuki, S., Harkin, D. G., Chirila, T. V. Incorporation of exogenous RGD peptide and inter-species blending as strategies for enhancing human corneal limbal epithelial cell growth on Bombyx mori silk fibroin membranes. J Funct Biomater. 2013;4(2):74-88.
58. Minoura N. AS, Gotoh Y., Tsukada M., Imai Y. Attachment and growth of cultured fibroblast cells on silk protein matrices. Journal of Biomedical Materials Research. 1995;29(10):1215-1221.
59. Gotoh Y, Tsukada, M., Minoura, N. Effect of the chemical modification of the arginyl residue in Bombyx mori silk fibroin on the attachment and growth of fibroblast cells. J Biomed Mater Res. 1998;39(3):351-357.
60. Minoura N, Aiba, S., Higuchi, M., Gotoh, Y., Tsukada, M., Imai, Y. Attachment and growth of fibroblast cells on silk fibroin. Biochemical and biophysical research communications. 1995;208(2):511-516.
61. Rockwood DN, Preda, R. C., Yucel, T., Wang, X., Lovett, M. L., Kaplan, D. L. Materials fabrication from Bombyx mori silk fibroin. Nat. Protocols. 2011;6(10):1612-1631.
62. Peterson MD, Mooseker, M. S. Characterization of the enterocyte-like brush border cytoskeleton of the C2BBe clones of the human intestinal cell line, Caco-2. Journal of Cell Science. 1992;102(3):581-600.
63. Ghio AJ, Dailey, L. A., Soukup, J. M., Stonehuerner, J., Richards, J. H., Devlin, R. B. Growth of human bronchial epithelial cells at an air-liquid interface alters the response to particle exposure. Particle and Fibre Toxicology. 2013;10(1):25.
64. Schamberger AC, Staab-Weijnitz, C. A., Mise-Racek, N., Eickelberg, O. Cigarette smoke alters primary human bronchial epithelial cell differentiation at the air-liquid interface. 2015;5:8163.
65. Stewart CE, Torr, E. E., Mohd Jamili, N. H., Bosquillon, C., Sayers, I. Evaluation of differentiated human bronchial epithelial cell culture systems for asthma research. Journal of Allergy. 2012;2012:11.
66. Ren H, Birch, N. P., Suresh, V. An optimised human cell culture model for alveolar epithelial transport. PLOS ONE. 2016;11(10):e0165225.
67. Fulcher ML, Gabriel, S., Burns, K. A., Yankaskas, J. R., Randell, S. H. Well-differentiated human airway epithelial cell cultures. Methods in molecular medicine. 2005;107:183-206.