臺灣博碩士論文加值系統

　　控制細胞在空間上的排列在細胞生物學、生物工程以及發育生物學等各種研究領域中是非常重要。例如神經或血管細胞只有當他們排列成特定的幾何形狀時才能形成具有功能性的組織或器官。
　　本文中，我們提出一個方法：它結合黃光微影，電濕式化學蝕刻以及溶劑鑄造技術來製備具有奈米級結構表面的甲殼素基板。我們利用哺乳動物細胞（例如，MDCK細胞和HeLa 細胞）來做測試發現細胞會感受我們基板的表面形貌並且對不同的表面形貌做出不同的反應。接著，我們也運用此細胞特性來達成微米級細胞圖形（例如:三角形、方形、圓形、愛心形和英文字母）排列的功能。此一具有生物相容性和生物降解性的甲殼素基板提供研究人員另一種可靠的細胞圖案化方法而可預期此甲殼素基板在生物醫學相關的應用中具有極大潛力。

The ability to control the spatial organization of single cells is critical in biomedically-relevant applications, and has provided tremendous insights in various research communities such as cell biology, bioengineering, as well as developmental biology. Functional nerves or blood vessels, for example, only can be formed when groups of cells are organized and aligned into specific geometries (or patterns).
Here we proposed a method, which is a combination of photolithography, electrochemical etching and solvent-casting technique, to prepare nanotexture-micropattern chitosan substrates. We demonstrated that mammalian cells (e.g., MDCK cells and HeLa cells) would sense the surface topographies of our substrates and form our designed patterns with micrometer-scale features (e.g., triangle, square, circular, heart and English letters). Our biocompatible and biodegradable chitosan substrates provide researchers with reliable alternative method of cell micropatterning and have a great potential in biomedical-relevant applications.

Acknowledgement………………………………………..…………………….…...Ⅰ
中文摘要………………………………………………………………………….….ⅡAbstract………………………………….……..…………………………….……...Ⅲ
Table of Contents.………………………………………………..……..……...........Ⅳ
List of Figures…………………………………………………………….…………Ⅷ
List of Tables………………………………………………………………….…Ⅻ
ChapterⅠIntroduction………………………………………..……………………..1
1.1 Background……………………………………………………..……………1
1.2 Method for Cell Micropatterning…………………………………….…….4
1.2.1 Magnetic Spheres…………………………………………...…….…..5
1.2.2 Dielectrophoresis…………………………………………...…..……..6
1.2.3 Photolithography…………………………………………...……....…8
1.2.4 Microcontact Printing……………………………………………..…10
1.3 Introduction to Chitosan…………………………………………….……....12
1.3.1 Origin and Properties………………………………………………..13
1.3.2 Chitosan for Cell Culture Substrates…………………………….…..15
1.3.3 Chitosan Substrates for Cell Micropatterning………………..….…..16
1.4 Objectives…………………………………………………………………...18
Chapter II Experimental…………………………………….………………..……20
2.1 Fabrication of Chitosan Substrates…………………………………….……20
2.1.1 Preparation of Chitosan Substrates from Different Molecular Weight
Powders…….…...………………………………………….………...……20
2.1.2 Preparation of Patterned Chitosan Substrates……………………..21
2.2 Characteristics of Chitosan Substrates………………………….…………..24
2.2.1 Scanning Electron Microscope……………………………………....24
2.2.2 Atomic Force Microscope………………………………...…...…….24
2.2.3 Contact Angle Meter………………………………………………...25
2.3 Cell Culture and Staining………………………...…………………………26
2.3.1 Cell Culture……………………………………...…………………..26
2.3.2 Cell Staining……………………………………...………………….26
2.3.3 Statistical Analysis…………………….………………………….....27
Chapter Ⅲ Results and Discussion…………………………………..…...………28
3.1 Characteristics of Chitosan Substrates Made from Different Molecular
Weight Powders…...………………………….…………………………………28
3.1.1 Top-view and Cross-section Topography of Chitosan Substrates…..28
3.1.2 Stiffness of Chitosan Substrates………………………………..……29
3.2 Surface Characteristics of Flat and Nanotextured Chitosan Substrates….…30
3.3 Cell Response to Flat and Nanotextured Chitosan Substrates …………..…31
3.4 Cell Micropatterning on Patterned Chitosan Substrates……………………34
3.5 Recyclable Chitosan Substrates……………………………………….……40
Chapter Ⅳ Conclusion…………………………………………...…………….….42
Chapter Ⅴ Future Work…………………………………………..………...……43
Reference………………………………………………...…………………………..48
Appendix A Cell Morphology, Proliferation, and Actin Organization on Functionalized Chitosan Substrates……………………………………………….54
A.1 Fabrication of Functionalized Chitosan Substrates………………………...54
A.1.1 Preparation of Chitosan Substrates with Different Functional Groups……………………………………………………………….…….54
A.1.2 Preparation of Chitosan Substrates with Nanotextured Surface…... 55
A.2 Cell Culture and Staining………………………………………………..…56
A.2.1 Cell Culture……………………………………………………..…..56
A.2.2 Cell Staining……………………..…………………………….……57
A.2.3 Statistical Analysis………………………………………….………57
A.3 Surface Characteristics of Functionalized Chitosan Substrates………..…..58
A.4 Cell Response on Functionalized Chitosan Substrates……………….…….60

1. C. S. Chen, M. Mrksich, S. Huang, G. M. Whitesides, D. E. Ingber, Science 1997, 276, 1425.
2. K. A. Kilian, B. Bugarija, B. T. Lahn, M. Mrksi, Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 4872.
3. C. Y. Fu, C. Y. Lin, W. C. Chu, H. Y. Chang, Tissue Eng. C 2011, 17, 871.
4. C. T. Ho, R. Z. Lin, W. Y. Chang, H. Y. Chang, C. H. Liu, Lab Chip 2006, 6, 724.
5. J. Y. Lee, S. S. Shah, C. C. Zimmer, G. Y. Liu, A. Revzin, Langmuir 2008, 24, 2232.
6. W. F. Liu, C. S. Chen, Materials Today 2005, 8, 28.
7. M. Mrksich, L. E. Dike, J. Tien, D. E. Ingber, G. M. Whitesides, Exp. Cell Res. 1997, 235, 305.
8. G. R. Ragetly, D. J. Griffon, H. B. Lee, L. P. Fredericks, W. G. Evans, Y. S. Chung, Acta Biomater. 2010, 6, 1430.
9. S. S. Silva, B. J. Goodfellow, J. Benesch, J. Rocha, J. F. Mano, R. L. Reis, Carbohydrate Polym. 2007, 70, 25
10. F. L. Mi, S. S. Shyu, Y. B. Wu, S. T. Lee, J. Y. Shyong, R. N. Huang, Biomaterials 2001, 22, 165.
11. Y. Kato, H. Onishi, Y. Machida, Biomaterials 2004, 25, 907.
12. S. V. Madihally, H. W. Matthew, Biomaterials 1999, 20, 1133.
13. K. T. Shalumon, K. H. Anulekha, C. M. Girish, R. Prasanth, S. V. Nair, R. Jayakumar, Carbohydrate Polym. 2010, 80, 413.
14. Y. Z. Zhang, B. Su, S. Ramakrishna, C. T. Lim, Biomacromolecules 2008, 9, 136.
15. Y. Z. Zhang, J. R. Venugopal, A. El-Turki, S. Ramakrishna, B. Su, C. T. Lim, Biomaterials 2008, 29, 4314.
16. Y. Zhou, D. Yang, X. Chen, Q. Xu, F. Lu, J. Nie, Biomacromolecules 2008, 9, 349.
17. Z. Q. Feng, X. Chu, N. P. Huang, T. Wang, Y. Wang, X. Shi, Y. Ding, Z. Z. Gu, Biomaterials 2009, 30, 2753.
18. R. Jin, L. S. Moreira Teixeira, P. J. Dijkstra, M. Karperien, C. A. van Blitterswijk, Z. Y. Zhong, J. Feijen, Biomaterials 2009, 30, 2544.
19. N. D. Leipzig, R. G. Wylie, H. Kim, M. S. Shoichet, Biomaterials 2011, 32, 57.
20. K. E. Crompton, J. D. Goud, R. V. Bellamkonda, T. R. Gengenbach, D. I. Finkelstein, M. K. Horne, J.S. Forsythe, Biomaterials 2007, 28, 441.
21. T. Hao, N. Wen, J. K. Cao, H. B. Wang, S. H. Lü, T. Liu, Q. X. Lin, C. M. Duan, C. Y. Wang, Osteoarthr. Cartil. 2010, 18, 257.
22. S. S. Silva, M. I. Santos, O. P. Coutinho, J. F. Mano, R. L. Reis, J. Mater. Sci. Mater. Med. 2005, 16, 575.
23. M. Cheng, J. Deng, F. Yang, Y. Gong, N. Zhao, X. Zhang, Biomaterials 2003, 24, 2871.
24. C. Mingyu, G. Kai, L. Jiamou, G. Yandao, Z. Nanming, Z. Xiufang, J. Biomater. Appl. 2004, 19, 59.
25. W. Cao, M. Cheng, Q. Ao, Y. Gong, N. Zhao, X. Zhang, J. Biomater. Sci. Polym. Ed. 2005, 16, 791.
26. T. Freier, H. S. Koh, K. Kazazian, M. S. Shoichet, Biomaterials 2005, 26, 5872.
27. S. Kim, M. E. Nimni, Z. Yang, B. Han, J. Biomed. Mater. Res. B 2005, 75, 442.
28. M. Peter, N. S. Binulal, S. Soumya, S. V. Nair, T. Furuike, H. Tamura, R. Jayakumar, Carbohydrate Polym. 2010, 79, 284.
29. M. Prabaharan, R. Jayakumar, Int. J. Biol. Macromol. 2009, 44, 320.
30. T. W. Chung, J. Yang, T. Akaike, K. Y. Cho, J. W. Nah, S. I. Kim, C. S. Cho, Biomaterials 2002, 23, 2827.
31. Y. Huang, S. Onyeri, M. Siewe, A. Moshfeghian, S.V. Madihally, Biomaterials 2005, 26, 7616.
32. A. Portero, D. Teijeiro-Osorio, M. J. Alonso, C. Remuñán-López, Carbohydrate Polym. 2007, 68, 617.
33. S. Itoh, I. Yamaguchi, M. Suzuki, S. Ichinose, K. Takakuda, H. Kobayashi, K. Shinomiyag, J. Tanaka, Brain Res. 2003, 993, 111.
34. R. Jayakumar, M. Prabaharan, P. T. Sudheesh Kumar, S. V. Nair, H. Tamura, Biotechnol. Adv. 2011, 29, 322.
35. P. R. Marreco, P. da Luz Moreira, S. C. Genari, A. M. Moraes, J. Biomed. Mater. Res. B 2004, 71, 268.
36. A. K. Azad, N. Sermsintham, S. Chandrkrachang, W. F. Stevens, J. Biomed. Mater. Res. B 2004, 69, 216.
37. C. A. Custódio, C. M. Alves, R. L. Reis, J. F. Mano, J. Tissue Eng. Regen. Med. 2010, 4, 316.
38. I. F. Amaral, R. E. Unger, S. Fuchs, A. M. Mendonca, S. R. Sousa, M. A. Barbosa, A. P. Pêgoa, C. J. Kirkpatrick, Biomaterials 2009, 30, 5465.
39. R. S. Tigli, M. Gumusderelioglu, Int. J. Biol. Macromol. 2008, 43, 121.
40. W. B. Tsai, Y. R. Chen, H. L. Liu, J. Y. Lai, Carbohydrate Polym. 2011, 85, 129.
41. D. Y. Wang, Y. C. Huang, H. Chiang, A. M. Wo, Y. Y. Huang, J. Biomed. Mater. Res. B 2007, 80, 447.
42. S. M. Luna, S. S. Silva, M. E. Gomes, J. F. Mano, R. L. Reis, J. Biomater. Appl. 2011, 26, 101.
43. J. G. Fernandez, C. A. Mills, E. Martinez, M. J. Lopez-Bosque, X. Sisquella, A. Errachid, J. Samitie, J. Biomed. Mater. Res. A 2008, 85, 242.
44. S. S. Silva, S. M. Luna, M. E. Gomes, J. Benesch, I. Pashkuleva, J. F. Mano, R. L. Reis, Macromol. Biosci. 2008, 8, 568.
45. Y. C. Wu, T. M. Lee, J. C. Lin, S. Y. Shaw, C. Y. Yang, J. Biomater. Sci. Polym. Ed. 2010, 21, 563.
46. N. Bhattarai, D. Edmondson, O. Veiseh, F. A. Matsen, M. Zhang, Biomaterials 2005, 26, 6176.
47. Y. Yuan, P. Zhang, Y. Yang, X. Wang, X. Gu, Biomaterials 2004, 25, 4273.
48. H. S. Liu, C. Y. Chen, C. H. Lee, Y. I. Chou, Br. J. Cancer 1998, 77, 1777.
49. C. Y. Yang, L. Y. Huang, T. L. Shen, J. A. Yeh, Eur. Cell Mater. 2010, 20, 415.
50. C. M. Cheng, P. R. LeDuc, Adv. Mater. 2008, 20, 953.
51. M. Pollock, Curr. Opin. Neurol. 1995, 8, 354.
52. D. Falconnet, G. Csucs, H. M. Grandin, M. Textor, Biomaterials 2006, 27, 3044.
53. C. S. Chen, M. Mrksich, S. Huang, G. M. Whitesides, D. E. Ingber, Science 1997, 276 1425.
54. R. Singhvi, A. Kumar, G. P. Lopez, G. N. Stephanopoulos, D. I. Wang, G. M. Whitesides, D. E. Ingber, Science 1994, 264, 696.
55. K. K. Parker, A. L. Brock, C. Brangwynne, R. J. Mannix, N. Wang, E. Ostuni, N. A. Geisse, J. C. Adams, G. M. Whitesides, D. E. Ingber, FASEB J. 2002, 16, 1195.
56. C. S. Chen, M. Mrksich, S. Huang, G. M. Whitesides, D. E. Ingber, Biotechnol. Progr. 1998, 14, 356.
57. R. S. Kane, S. Takayama, E. Ostuni, D. E. Ingber, G. M. Whitesides, Biomaterials 1999, 20, 2363.
58. Y. Xia, G. M. Whitesides, Angew. Chem. Int. Ed. 1998, 37, 550.
59. R. D. Piner, J. Zhu, F. Xu, S. Hong, C. A. Mirkin, Science 1999, 283, 661.
60. K. Y. Suh, J. Seong, A. Khademhosseini, P. E. Laibinis, R. A. Langer, Biomaterials 2004, 25, 557.
61. R. S. Kane, S. Takayama, E. Ostuni, D. E. Ingber, G. M.Whitesides, Biomaterials 1999, 20, 2363.
62. G. M. Whitesides, E. Ostuni, S. Takayama, X. Jiang, D. E. Ingber, Annu. Rev. Biomed. Eng. 2001, 3, 335.
63. K. Salaita, Y. Wang, C. A. Mirkin, Nat. Nanotechnol. 2007, 2, 145.
64. E. A. Roth, T. Xu, M. Das, C. Gregory, J. J. Hickman, T. Boland, Biomaterials 2004, 25, 3707.
65. J. S. Miller, M. I. Bethencourt, M. Hahn, T. R. Lee, J. L. West, Biotechnol. Bioeng. 2006, 93, 1060.
66. G. Kumar, Y. C. Wang, C. Co, C. C. Ho, Langmuir 2003, 19, 10550.
67. Y. C. Wang, C. C. HO, FASEB J. 2004, 18, 525.
68. C. C. Co, Y. C. Wang, C. C. Ho, J. Am. Chem. Soc. 2005, 127, 1598.
69. C. Y. Yang, T. C. Liao, H. H. Shuai, T. L. Shen, J. A. Yeh, C. M. Cheng, Biomaterials 2012, 33, 4988.
70. R. N. Wenzel, Ind. Eng. Chem 1936, 28, 988.
71. A. W. Adamson, A. P. Gast, Physical Chemistry of Surface, 6th ed., Wiley, New York, 1997.
72. A. Curtis, C. Wilkinson, Biomaterials 1997, 18, 1573.