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

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