細胞層片技術被廣泛的應用於修補受損的器官，例如眼睛、心臟與膀胱，
然而，細胞層片技術主要的難題在於從培養容器分離細胞時，細胞層片會因為
激動蛋白絲產生都收縮力而皺縮與摺疊，因此需要一個能夠維持細胞層片構
型，並且具有良好生物相容性的素材支持細胞層片。明膠甲基丙烯水凝膠
(GelMa)由於其優良的生物相容性與易塑型的特性而足以作為培養細胞層片的
媒介，本研究使用明膠甲基丙烯水凝膠培養細胞層片，並且探討將所建構細胞
層片應用於生物醫學領域的可能性，包含傷口癒合試驗與腫瘤與正常細胞共培
養試驗。於傷口癒合試驗中，測試了單磷酸核苷酸 AMP、TMP、GMP、CMP 以及四種單磷酸合苷酸混合液對傷口癒合的影響，結果顯示 AMP 與 GMP 對
傷口癒合有抑制的效果；於腫瘤與正常細胞共培養試驗中，發現由較少細胞所
組成的多細胞類球體於小鼠纖維母細胞 3T3 組成的細胞層片有較好的生長與 擴散。接著成功的以甲基丙烯水凝膠與人類腸道上皮細胞 Int-407 培養於空氣 -液體介面(Air-liquid interface)培養系統，所培養的細胞經過 7 天的空氣-液體
介面培養後仍保持細胞活性，而後進行傷口癒合試驗，測試單磷酸合苷酸在空
氣-液體介面培養系統如何影響傷口癒合。

Cell sheet technology is widely used to repair damaged organs such as eyes, heart and bladder. However, the main challenge of cell sheet technology is that, when separating the cells sheet from the culture vessel, the cell sheet shrinks due to the contractile force produced by actin filaments pulling neighboring cells together. Thus, a supporting material that maintains the cell layer configuration with good biocompatibility is needed. Gelatin methacryloyl (GelMa) hydrogels is sufficient as a medium for culturing cell sheet due to its excellent biocompatibility and tunability. This study uses GelMa hydrogels to culture cell sheet, and explores the possibility of utilizing the cell sheet in the biomedical field, including wound healing tests and tumor-cell co-culture experiments. In the wound healing test, the effects of AMP, TMP, GMP, CMP and the mixture of four monophosphate nucleosides on wound healing were tested. The results showed that AMP and GMP have an inhibitory effect on wound healing. In the tumor-cell co-culture experiments, it was found that the multicellular spheroids composed of fewer cells had better growth and spread in the cell sheet composed of mouse fibroblasts 3T3. The GelMa hydrogel with human intestinal epithelial cells Int-407 were successfully cultured in an air-liquid interface culture system, and the cultured cells were maintained after 7 days on air-liquid interface culture system with good cell viability. A wound healing test was performed to test how monophosphate nucleoside affects wound healing on the air-liquid interface culture system.

中文摘要........................................................................................................................ 1
Abstract .......................................................................................................................... 2
致謝................................................................................................................................ 3
縮寫表............................................................................................................................ 5
目錄................................................................................................................................ 6
圖目錄............................................................................................................................ 7
前言................................................................................................................................ 8
材料與方法........................................................................................................ 13
1.1 細胞培養....................................................................................................... 13
1.2 甲基丙烯酸化明膠的製備........................................................................... 14
1.3 傷口癒合試驗............................................................................................... 15
1.4 腫瘤細胞與小鼠纖維母細胞3T3 共培養試驗 .......................................... 16
1.5 多細胞類球體與小鼠纖維母細胞3T3 共培養試驗 .................................. 17
1.6 空氣-液體介面培養系統的建構 ................................................................. 18
1.7 免疫螢光染色............................................................................................... 18
1.8 空氣-液體介面培養的傷口癒合試驗 ......................................................... 19
結果.................................................................................................................... 21
2.1 小鼠纖維母細胞3T3 培養於明膠甲基丙烯水凝膠 .................................. 21
2.2 小鼠纖維母細胞3T3 於明膠甲基丙烯水凝膠上之傷口癒合試驗 .......... 21
2.3 腫瘤細胞與小鼠纖維母細胞3T3 於明膠甲基丙烯水凝膠之共培養試驗
.............................................................................................................................. 23
2.4 多細胞類球體與小鼠纖維母細胞3T3 於明膠甲基丙烯水凝膠之共培養
試驗...................................................................................................................... 23
2.5 空氣-液體介面培養系統的建構 ................................................................. 24
2.6 人類腸道上皮細胞Int-407 於空氣-液體介面培養系統的傷口癒合試驗 25
討論.................................................................................................................... 26
參考文獻............................................................................................................ 29

1. Nishida, K., et al., Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med, 2004. 351(12): p. 1187-96.
2. Masuda, S., et al., Cell sheet engineering for heart tissue repair. Adv Drug Deliv Rev, 2008. 60(2): p. 277-85.
3. Yamato, M. and T. Okano, Cell sheet engineering. Materials Today, 2004. 7(5): p. 42-47.
4. Tang, Z., Y. Akiyama, and T. Okano, Temperature-Responsive Polymer Modified Surface for Cell Sheet Engineering. Vol. 4. 2012. 1478-1498.
5. Shimizu, T., et al., Two-dimensional manipulation of cardiac myocyte sheets utilizing temperature-responsive culture dishes augments the pulsatile amplitude. Tissue Eng, 2001. 7(2): p. 141-51.
6. Wongin, S., et al., Effect of Cell Sheet Manipulation Techniques on the Expression of Collagen Type II and Stress Fiber Formation in Human Chondrocyte Sheets. Tissue Eng Part A, 2018. 24(5-6): p. 469-478.
7. Annabi, N., et al., 25th anniversary article: Rational design and applications of hydrogels in regenerative medicine. Advanced materials (Deerfield Beach, Fla.), 2014. 26(1): p. 85-123.
8. Thiele, J., et al., 25th anniversary article: Designer hydrogels for cell cultures: a materials selection guide. Adv Mater, 2014. 26(1): p. 125-47.
9. Alge, D.L. and K.S. Anseth, Bioactive hydrogels: Lighting the way. Nat Mater, 2013. 12(11): p. 950-2.
10. Hunt, N.C., et al., 3D culture of human pluripotent stem cells in RGD-alginate hydrogel improves retinal tissue development. Acta Biomater, 2017. 49: p. 329-343.
11. Short, A.R., et al., Imaging Cell-Matrix Interactions in 3D Collagen Hydrogel Culture Systems. Macromol Biosci, 2017. 17(6).
12. Dosh, R.H., et al., Use of hydrogel scaffolds to develop an in vitro 3D culture model of human intestinal epithelium. Acta Biomater, 2017. 62: p. 128-143.
13. Luo, Y. and M.S. Shoichet, A photolabile hydrogel for guided three-dimensional cell growth and migration. Nat Mater, 2004. 3(4): p. 249-53.
14. West, J.L., Protein-patterned hydrogels: Customized cell microenvironments. Nat Mater, 2011. 10(10): p. 727-9.
15. Tibbitt, M.W. and K.S. Anseth, Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnology and bioengineering, 2009. 103(4): p. 655-663.
16. Chen, P., et al., Intra-articular delivery of sinomenium encapsulated by chitosan microspheres and photo-crosslinked GelMA hydrogel ameliorates osteoarthritis by effectively regulating autophagy. Biomaterials, 2016. 81: p. 1-13.
17. Modaresifar, K., A. Hadjizadeh, and H. Niknejad, Design and fabrication of GelMA/chitosan nanoparticles composite hydrogel for angiogenic growth factor delivery. Artif Cells Nanomed Biotechnol, 2018. 46(8): p. 1799-1808.
18. Wang, Y., et al., Development of a Photo-Crosslinking, Biodegradable GelMA/PEGDA Hydrogel for Guided Bone Regeneration Materials. Materials (Basel), 2018. 11(8).
19. Liu, Y. and M.B. Chan-Park, A biomimetic hydrogel based on methacrylated dextran-graft-lysine and gelatin for 3D smooth muscle cell culture. Biomaterials, 2010. 31(6): p. 1158-70.
20. Van den Steen, P.E., et al., Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9). Crit Rev Biochem Mol Biol, 2002. 37(6): p. 375-536.
21. Qi, H., et al., Patterned differentiation of individual embryoid bodies in spatially organized 3D hybrid microgels. Adv Mater, 2010. 22(46): p. 5276-81.
22. Bertassoni, L.E., et al., Hydrogel bioprinted microchannel networks for vascularization of tissue engineering constructs. Lab Chip, 2014. 14(13): p. 2202-11.
23. Yin, J., et al., 3D Bioprinting of Low-Concentration Cell-Laden Gelatin Methacrylate (GelMA) Bioinks with a Two-Step Cross-linking Strategy. ACS Appl Mater Interfaces, 2018. 10(8): p. 6849-6857.
24. Zamanian, B., et al., Interface-directed self-assembly of cell-laden microgels. Small, 2010. 6(8): p. 937-44.
25. Annabi, N., et al., Hydrogel-coated microfluidic channels for cardiomyocyte culture. Lab Chip, 2013. 13(18): p. 3569-77.
26. Cha, C., et al., Microfluidics-assisted fabrication of gelatin-silica core-shell microgels for injectable tissue constructs. Biomacromolecules, 2014. 15(1): p. 283-90.
27. Krunkosky, T.M., et al., Mycoplasma pneumoniae host-pathogen studies in an air-liquid culture of differentiated human airway epithelial cells. Microb Pathog, 2007. 42(2-3): p. 98-103.
28. Chen, C.-L., et al., The apical-basal cell polarity determinant Crumbs regulates Hippo signaling in Drosophila. Proceedings of the National Academy of Sciences, 2010. 107(36): p. 15810-15815.
29. Martin-Belmonte, F. and K. Mostov, Regulation of cell polarity during epithelial morphogenesis. Curr Opin Cell Biol, 2008. 20(2): p. 227-34.
30. Roignot, J., X. Peng, and K. Mostov, Polarity in mammalian epithelial morphogenesis. Cold Spring Harb Perspect Biol, 2013. 5(2).
31. Whitcutt, M.J., K.B. Adler, and R. Wu, A biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells. In Vitro Cell Dev Biol, 1988. 24(5): p. 420-8.
32. Pezzulo, A.A., et al., The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia. Am J Physiol Lung Cell Mol Physiol, 2011. 300(1): p. L25-31.
33. de Jong, P.M., et al., Ciliogenesis in human bronchial epithelial cells cultured at the air-liquid interface. Am J Respir Cell Mol Biol, 1994. 10(3): p. 271-7.
34. Ootani, A., et al., An air-liquid interface promotes the differentiation of gastric surface mucous cells (GSM06) in culture. Biochem Biophys Res Commun, 2000. 271(3): p. 741-6.
35. Karam, S.M., Dynamics of epithelial cells in the corpus of the mouse stomach. IV. Bidirectional migration of parietal cells ending in their gradual degeneration and loss. Anat Rec, 1993. 236(2): p. 314-32.
36. Nossol, C., et al., Air-liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC). Histochem Cell Biol, 2011. 136(1): p. 103-15.
37. Bradbury, M.W.B., Introduction to the Blood-Brain Barrier. Methodology, Biology and Pathology. Journal of Anatomy, 1999. 194(Pt 1): p. 153-157.
38. Li, J., et al., Pseudomonas aeruginosa Exoprotein-Induced Barrier Disruption Correlates With Elastase Activity and Marks Chronic Rhinosinusitis Severity. Frontiers in Cellular and Infection Microbiology, 2019. 9(38).
39. Movia, D., et al., Multilayered Cultures of NSCLC cells grown at the Air-Liquid Interface allow the efficacy testing of inhaled anti-cancer drugs. Sci Rep, 2018. 8(1): p. 12920.
40. Ong, H.X., et al., Primary Air-Liquid Interface Culture of Nasal Epithelium for Nasal Drug Delivery. Mol Pharm, 2016. 13(7): p. 2242-52.
41. Lin, R.Z., et al., Tumor-induced endothelial cell apoptosis: roles of NAD(P)H oxidase-derived reactive oxygen species. J Cell Physiol, 2011. 226(7): p. 1750-62.
42. Achilli, T.M., J. Meyer, and J.R. Morgan, Advances in the formation, use and understanding of multi-cellular spheroids. Expert Opin Biol Ther, 2012. 12(10): p. 1347-60.
43. Dunn, L., et al., Murine model of wound healing. J Vis Exp, 2013(75): p. e50265.
44. Planz, V., J. Wang, and M. Windbergs, Establishment of a cell-based wound healing assay for bio-relevant testing of wound therapeutics. J Pharmacol Toxicol Methods, 2018. 89: p. 19-25.
45. Gendaszewska-Darmach, E. and M. Kucharska, Nucleotide receptors as targets in the pharmacological enhancement of dermal wound healing. Purinergic Signal, 2011. 7(2): p. 193-206.
46. Wang, J., et al., Intracellular adenosine triphosphate delivery enhanced skin wound healing in rabbits. Ann Plast Surg, 2009. 62(2): p. 180-6.
47. Langemo, D.K., et al., Two-dimensional wound measurement: comparison of 4 techniques. Adv Wound Care, 1998. 11(7): p. 337-43.
48. Ramos Leal, G., et al., Role of cAMP modulator supplementations during oocyte in vitro maturation in domestic animals. Anim Reprod Sci, 2018. 199: p. 1-14.
49. Boularan, C. and C. Gales, Cardiac cAMP: production, hydrolysis, modulation and detection. Front Pharmacol, 2015. 6: p. 203.
50. Williams, J.P., Interrelation of epithelial glycogen, cell proliferation and cellular migration with cyclic adenosine monophosphate in epithelial wound healing. Cell Differ, 1972. 1(5): p. 317-23.
51. Schwarz, K.R.L., et al., The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation. PLoS One, 2018. 13(1): p. e0191023.
52. Lopes-Pires, M.E., et al., PKC and AKT Modulate cGMP/PKG Signaling Pathway on Platelet Aggregation in Experimental Sepsis. PLoS One, 2015. 10(9): p. e0137901.
53. Fallahian, F., et al., Cyclic GMP induced apoptosis via protein kinase G in oestrogen receptor-positive and -negative breast cancer cell lines. Febs j, 2011. 278(18): p. 3360-9.
54. Greijer, A.E. and E. van der Wall, The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis. Journal of clinical pathology, 2004. 57(10): p. 1009-1014.
55. Qiu, Y., P. Li, and C. Ji, Cell Death Conversion under Hypoxic Condition in Tumor Development and Therapy. International journal of molecular sciences, 2015. 16(10): p. 25536-25551.
56. Yue, K., et al., Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. Biomaterials, 2015. 73: p. 254-271.