微圖型化甲殼素基材用於神經元導引之發展｜國立清華大學博碩士論文庫

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研究生：	宋淳諺 Sung, Chun-Yen
論文名稱：	微圖型化甲殼素基材用於神經元導引之發展 Development of Micropatterned Chitosan Substrates for Neuronal Guidance
指導教授：	葉哲良 Yeh, J.Andrew 鄭兆珉 Cheng, Chao-Min
口試委員:	陳文翔 Chen, Wen-Shiang 王仰高 Wang, Yang-Kao
學位類別：	碩士 Master
系所名稱：	工學院 - 奈米工程與微系統研究所 Institute of NanoEngineering and MicroSystems
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	85
中文關鍵詞：	甲殼素基材、黃光微影、感應耦合式電漿反應離子蝕刻、濕式化學蝕刻、溶劑鑄造技術
外文關鍵詞：	Chitosan substrates, Neuro-2a cells, ICP-RIE, wet chemical etching, solvent casting
相關次數：	點閱：3 下載：0
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本篇論文設計出微圖型化表面之甲殼素基材排列神經母細胞株Neuro-2a，希望在基材上建立神經元連結網路。我們使用微奈米技術，如黃光微影、感應耦合式電漿反應離子蝕刻、濕式化學蝕刻和溶劑鑄造技術來製備六種具奈米結構圖案的甲殼素基材。
首先，我們探討神經母細胞株Neuro-2a和甲殼素基材（平坦和全奈米結構）之間的相互作用關係。我們發現神經母細胞株Neuro-2a生長在平坦甲殼素基材其攤附面積大於在奈米結構甲殼素基材，且易於在平坦區攤附和生長。其次，我們為了在甲殼素基材上建立神經元連結網路，包含細胞本體排列和神經突觸導引，我們發展了六種型式的微圖型化甲殼素基材，包含矩形圖案、單一細胞圖案、線圖案、負片甲殼素基材、網狀圖案和極化圖案。研究中發現極化圖案是目前建立神經元連結網路最有效果之甲殼素基材。其原理先用表面形貌控制神經細胞極化方向，再設計溝槽方向符合神經突觸之分化方向，導引樹突和軸突連結。此研究將有潛力成為探討神經科學的平台，如神經傳導物質的測試和電生理刺激平台之應用，相信對生物醫學工程將是一大貢獻。

This thesis describes micropatterning of Neuro-2a cell lines on modified chitosan substrates with microstructural pattern made through nanofabrication approaches to form neuronal networks. We performed methods of fabrication which combined with photolithography, inductively coupled plasma reactive ion etching (ICP-RIE), wet chemical etching and solvent casting to prepare six different types of chitosan substrates.
Firstly, the neuronal cell-substrate interaction (flat and nanostructural) was investigated. We found that the projected cell area of Neuro-2a cells on flat chitosan substrates was larger than on nanostructural chitosan substrates and Neuro-2a cells preferred to adhere on flat chitosan surface region than on nanostructural chitosan substrates to immobilize and differentiation. Secondly, in order to grow neuronal network including somas patterning and neurites guidance, we have developed chitosan substrates, such as square pattern, single cell pattern, line pattern, negative chitosan substrates, network pattern and polarity induce pattern. We found that the polarity induce pattern was the most suitable approach to form neuronal network by controlling cell polarity in designed constrain geometry, and then, we designed grooves to fit the direction of neurites outgrowth. This research, we believe, would have the potential to study a wide range of neurobiological applications such as neurotransmitters screening and electrophysiological stimulation platforms. This development of chitosan-based platforms would contribute to biomedical engineering.

Contents
Acknowledgement    I
中文摘要    II
Abstract    III
Contents    IV
Captions of Figures    V
Tables    XIII

Chapter I Introduction    1
1.1 Background    1
1.2 Effect of Micro-and Nano-Topographic Surface on Neuronal Cells Morphology    4
1.2.1 Cell Adhesion    5
1.2.2 Cell Polarity    8
1.3 Methods for Neuronal Cells Patterning    12
1.3.1 Chemical Approach    12
1.3.2 Physical Approach    18
1.4 Introduction to Chitosan    19
1.4.1 Origin and Properties    19
1.4.2 Chitosan Substrate for Neuronal Cells Culture    21
1.5 Motivation and Objective    22
Chapter II Experiments    24
2.1 Experimental Processes    24
2.2 Fabrication of Chitosan Substrates    24
2.2.1 Evolution of Patterned Chitosan Substrates    24
2.2.2 Preparation of Patterned Chitosan Substrates    25
2.3 Neuronal Cells Culture    32
2.4 Cell Morphology and Cytoskeleton of Neuronal Cell    33
2.4.1 Scanning Electron Microscope    33
2.4.2 Cell Staining    34
2.5 Statistical Analysis    35
Chapter III Results and Discussion    36
3.1 Characteristics of Chitosan Substrates    36
3.2 Neuronal Cells Response to Flat and Nanostructural Chitosan Substrates    37
3.3 Neuronal Cells Micropatterning on Patterned Chitosan Substrates    42
3.3.1 Square Pattern    43
3.3.2 Single Cell Pattern    49
3.3.3 Line Pattern    50
3.3.4 Negative Chitosan    51
3.3.5 Network Pattern    55
3.3.6 Polarity Induce Pattern    59
Chapter IV Discussion    66
Chapter V Conclusion    72
Chapter VI Future Work    73
6.1 Live Imaging    73
6.2 Drug Screening    73
6.3 Gene Expression of Neuronal Cells on Chitosan Substrates    73
6.4 Patch Clamp    73
6.5 Mechanical Force Test Platform    74
References    75
Appendix A: Etch Process of Black Wafer Silicon    79
Appendix B: Protocol for Solvent Casting Process of Chitosan Substrates    81
Appendix C: Neuronal Cellular Morphology on Functionalized Chitosan Substrates    83

                                

References
[1] C. M. Loya; D. V. Vactor; T. A. Fulga. Genes and Development 2010, 24, 625.
[2] K. Fenrich; T. Gordon. Canadian Journal of Neurological Sciences 2004, 31, 142.
[3] W. W. Campbell. Clinical Neurophysiology 2008, 119, 1951.
[4] F. G. Giancotti; E. Ruoslahti. Science 1999, 285, 1028.
[5] B. Ladoux; A. Nicolas. Reports on Progress in Physics 2012, 75, 116601.
[6] B. Geiger; A. Bershadsky; R. Pankov; K. M. Yamada. Nature Reviews Molecular
Cell Biology 2001, 2, 793.
[7] B. R. Zaidel; M. Cohen; L. Addadi; B. Geiger. Biochemical Society Transactions 2004, 32, 416.
[8] N. Q. Balaban; U. S. Schwarz; D. Riveline; P. Goichberg; G. Tzur; I. Sabanay. Nature Cell Biology 2001, 3, 466.
[9] A. D. Bershadsky; C. Ballestrem; L. Carramusa; Y. Zilberman; B. Khochbin.
European Journal of Cell Biology 2006, 85, 165.
[10] B. Ladoux; A. Nicolas. Reports on Progress in Physics 2012,75, 116601.
[11] H. G. Craighead; S. W. Turner; R. C. Davis; C. James; A. M. Perez; P. M. St. John; M. S. Isaacson; L. Kam; W. Shain; J. N. Turner; G. Banker. Biomedical Microdevices 1998, 49.
[12] N. Li; A.Folch. Experimental Cell Research 2005, 311, 307.
[13] M.Cecchini; G.Bumma; M.Serresi; F.Beltram. Nanotechnology 2007, 18, 505103.
[14] A. Ferrari; M. Cecchini; M. Serresi; P. Faraci; D. Pisignano; F. Beltramal; Biomaterials 2010, 31, 4682.
[15] A. Béduer; C. Vieu; F. Arnauduc; J. C. Sol; I. Loubinoux; Laurence Vaysse. Biomaterials 2012, 33, 504.
[16] D. W. Branch; B. C. Wheeler. Transactions on Biomedical Engineering 2000, 47, 3.
[17] H. I. Wu; G. H. Cheng; Y. Y. Wong; C. M. Lin; W. Fang; W. Y. Chowde; Y. C. Chang. Lab on a Chip 2010, 10, 647.
[18] K. Stephan; W. Dertinger; X. Jiang; Z. Li; V. N. Murthy; G. M. Whitesides. Proceedings of the National Academy of Sciences of the United States of America 2002, 99, 12542.
[19] T. Esch; V. Lemmon; G. Banker. The Journal of Neuroscience 1999,19, 6417.
[20] K.E. Cromptona; J.D. Goudc; R.V. Bellamkondac; T.R. Gengenbachd; D.I. Finkelsteine; M.K. Hornef; J.S. Forsythe. Biomaterials 2007, 28, 441.
[21 ] S. B. Jun; M. R. Hynd; N. Dowell-Mesfin; K. L. Smith; James N. Turner; W. Shain; S. J. Kim. Journal of Neuroscience Methods 2007, 160, 317.
[22] I. Poudel; J. S. Lee; L. Tan; J. Y. Lim; Acta Biomaterialia 2013, 9, 4592.
[23] A. K. Vogt; L. Lauer; W. Knoll; O. Andreas. Biotechnology Progress 2003, 19, 1562.
[24] O. Andreas; B. M. Simone; D. Tanja; H. Rita; G. Peter; G. Jurgen; M. L. Martin ; R. Anna; S. Susanne; S. Petra; V. E. Angela. Soft matter 2007, 3, 290.
[25] D.W. Branch; J.M.Corey; J.A.Weyhenmeyer; G.J. Brewer; B.C. Wheeler. Medical and Biological Engineering and Computing 1998, 36, 135.
[26] J. C. Chang; G. J. Brewerb; B. C. Wheeler. Biomaterials 2003, 24, 2863.
[27] A. K. Vogt; W. Gunter; M. Wolfgang; K. Wolfgang. Biomaterials 2005, 26, 2549.
[28] L. Kam; W. Shain; J.N. Turner; R. Bizios. Biomaterials 2001, 22, 1049.
[29] C. Anne; M. Dolores; M. Robert; C. Tanya; M. Raluca; P. Christophe; D. Mike; K. Anthony; M. Geoff. Biotechnology and Bioengineering 2010, 105, 368.
[30] C. Wyart; C. Ybert; L. Bourdieu; C. Herr, C. Prinz; D. Chatenay. Journal of Neuroscience Methods 2002, 117, 123.
[31] J. P. Frimat; J. Sisnaiske; S. Subbiah; H. Menne; P. Godoy; P. Lampen; M. Leist; J. Franzke; J. G. Hengstler; C. Thrielx; J. Westx. Lab on a chip 2010,10, 701.
[32] J. Zhang; S. Venkataramani; H. Xu; Y. K. Song; H. K. Song; G. T. R. Palmore; J. Fallonc; A. V. Nurmikko. Biomaterials 2006, 27, 5734.
[33] C. Ji; Z.Geng; W. Lei; D. Xiaowei; Z. H; W. Bernhard; J. Qinghui; Z. Jianlong; O. Andreas. Yuansen Xua, Journal of Neuroscience Methods 2014,213,196.
[34] G. S. Withers; C. D. James; C. E. Kingman; H. G. Craighead; G. A. Banker. Journal of Neurobiology 2006, 66, 1183.
[35] J. M. Corey; E. L. Feldman. Experimental Neurology 2003, 184, 89.
[36 ] M. Merz; P. Fromherz. Advanced functional material 2005, 5, 739.
[37] M. Merz; P. Fromherz. Advanced material 2002, 14, 141.
[38] S. V. Madihally; H. W.T. Matthew. Biomaterials 1999, 20, 1133.
[39] T. Freiera; R. Montenegro; H. S. Koha; M. S. Shoichet. Biomaterials 2005, 26, 4624.
[40] H. M. K¨oping; Y.S. Mel’nikova; K.M. Vårum; B. Lindman; P. Artursson. The Journal of Gene Medicine 2003, 5, 130.
[41] I.Y. Kim; S.J. Seo; H.S. Moon; M.K. Yoo; I.Y. Park; B.C Kim; C.S. Cho. Biotechnology Advances 2008, 26, 1.
[42] Q. Ao; C.K. Fung; Y. Tsui. Biomaterials 2011, 32,787.
[43] M. Cheng; J. Deng; F.Yang; Y. Gong; N. Zhao; X. Zhang. Biomaterials 2003, 24, 2871.
[44] A. Wang; Q. Ao; W. Cao; M. Yu; Q. He; L. Kong; L. Zhang; Y. Gong; X. Zhang. Journal of Biomedical Materials Research Part A 2006, 36.
[45] H.-H. Shuai; C. Y. Yang; H. I.C. Harn; R. L. York; T. C. Liao; W. S. Chen, J. A. Yeh; C. M. Cheng. Chemical Science 2013.
[46] C. Max Hsieh, 國立清華大學碩士論文 2007.
[47] S. Y. Chou; C. M. Cheng; C. C. Chen; P. R. LeDuc. Soft Matter 2011, 7, 9871.

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