Investigation of Mechanical Tension on Neurite Outgrowth and Axon Elongation with Micropatterned Stretching System

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研究生：	張友儒 Chang, Yu-Ju
論文名稱：	Investigation of Mechanical Tension on Neurite Outgrowth and Axon Elongation with Micropatterned Stretching System 以微拉伸系統探討應力刺激及微圖案對神經突觸生長之研究
指導教授：	蔡哲正王子威
口試委員:	蔡哲正王子威孫瑞昇
學位類別：	碩士 Master
系所名稱：	工學院 - 材料科學工程學系 Materials Science and Engineering
論文出版年：	2011
畢業學年度：	100
語文別：	英文
論文頁數：	90
中文關鍵詞：	應力刺激、黃光微影、微流道、突觸生長、軸突延長
外文關鍵詞：	Mechanical tension, Photolithography, Microfluidic, Neurite extension, Axon elongation
相關次數：	點閱：1 下載：0
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Mechanical tension is regarded as a crucial factor to trigger neurite outgrowth and axon elongation in many neurological researches. In this study, we developed a feasible and reliable stretching platform combined with photolithography and microfluidic techniques to investigate the effect of tensile force on cell mechanics of neurons. The PDMS based substrate with specific micropattern is designed as stretching membrane where neural stem cells (NSCs) are seeded on and experienced with tensile force. This allows us to investigate the mechanoresponses of neuron and verify the effects of tension on neurite extension at a single-cell level. Different stretching mode (viz., continuous or intermittent) and culture conditions (viz., pattern or non-pattern) are conducted to examine the distinct effects of tension on neurite extension during cell incubation. For cells undergoing mechanical tension, neurite extension and axon elongation were significantly enhanced about 30% and neurite orientation was heading more toward the path of tension as cells experienced stretching with parameter 1 mm/day after 7 days culture. We can also find that mechanical tension apparently influence NSCs differentiation toward neuronal cells at Day 7 from the analysis of neuronal protein marker, β Ⅲ tubulin. However, the neuronal maturity didn’t show significant difference for neurites either on parallel or vertical channels. This may be further investigated by long-term culture to distinguish the directional effect of mechanical tension on promoting NSCs maturation processes.

在過去許多探討神經細胞的力學研究當中，應力刺激對神經突觸的生長，扮演著相當重要的角色。本篇研究裡，我們發展出一套實效且穩定的拉伸平台，並結合黃光微影的微流道技術，探討神經細胞的力學現象。我們主要是利用具有彈性的PDMS為拉伸的基材，經由黃光微影製程，在其上製作特定尺寸的微流道，經由拉伸PDMS基材，使培養在基材上的神經幹細胞可以感受到拉伸應力的刺激。這樣的設計，便於我們探討神經細胞的力學反應，並可觀察接近單一細胞尺寸之神經軸突的生長。經由不同的培養條件及拉伸參數測試，當神經幹細胞受到拉伸而感受到應力刺激時，神經突觸的生長，及軸突的延長有顯著增加的效果，以預先讓細胞貼附12小時，再進行拉伸實驗測試而言，拉伸參數為1 mm/day的組別在7天的培養之後，總突觸長度可達240 μm，比沒有拉伸的組別增加約30%，突觸相位角為較趨於水平方向的28°。此外，我們亦利用免疫螢光染色，觀察細胞在受到機械應力刺激後的分化成熟情況，從標定神經細胞的螢光蛋白β Ⅲ tubulin之表現量得知，當神經幹細胞受到拉伸應力刺激時，大約於培養7天後，比未受應力刺激的神經幹細胞，有明顯朝向神經細胞分化成熟的趨勢。而神經幹細胞在水平方向，和垂直微流道方向上的成熟差異性，以目前的實驗天數未觀察到有顯著的差別，需再經由長時間的培養觀察，才得以看出應力刺激，對水平和垂直於拉伸方向的神經突觸，其生長和分化成熟度的影響。

Table of Contents
Chapter 1 Introduction    1
1 Background Information    1
1.1 Cell Mechanics of Neuron    1
1.2 Methods to Investigate Neuron Mechanics    3
1.3 Microfluidics    3
2 Motivation and Purpose    5
Chapter 2 Literature Review    6
1 Fundamental Concepts of Neurology    6
1.1 Neuron    6
1.2 Neurite Growth    7
2 Mechanism of Tension Generated from Cytoskeletal Dynamics    9
3 Mechanical Tension in Axon Growth    13
3.1 Tip Growth    13
3.2 Stretch Growth    13
3.3 Force Order to Trigger Neurite Growth    14
4 Force Stimulation Device to Investigate Neuron Mechanics    15
4.1 Towing by Micromanipulator    15
4.2 Stretching by Rotary Translation Stage    16
4.3 Stretching by Linear Translation Stage    17
5 Fabrication of PDMS-based Micropatten by Photolithography    18
5.1 Microfluidics: An Overview    18
5.2 Mask Fabrication    20
5.3 Preparation of Wafer    20
5.4 Photoresist Spinning and Soft Bake    20
5.5 Exposure and Post-Exposure Treatment    21
5.6 Development, Descumming, and Post-Baking    23
5.7 Application of Mold Release Layer    23
5.8 PDMS Molding    23
5.9 Creation of PDMS Microfluidics    24
Chapter 3 Materials and Methods    25
1 Experimental Design    28
2 Preparation of Stretching Sample    30
2.1 Mask Design    30
2.2 Master Preparation    32
2.3 PDMS Molding    33
2.4 Reservoir Fabrication    34
2.5 Reservoir Adhesion    35
2.6 Laminin Coating    35
3 Characterization of Sample    36
3.1 Morphology Inspection by Optical Microscope    36
3.2 Cross-section Analysis by SEM    36
4 Cell Culture    37
4.1 Mouse 3T3 fibroblast    37
4.2 PC 12    37
4.3 Neural Stem Cells    37
5 Cytotoxicity Test    38
6 Stretching Test    39
6.1 Stretching Device    39
6.2 Control of Stretching Mode    42
6.3 Conduction of Stretching Experiments    42
7 Characterization of Cell Stretching    43
7.1 Morphological Performances    43
7.2 Immunocytochemistry    43
Chapter 4 Results    44
1 Master Examination    44
2 PDMS Molding    47
3 Cytotoxicity Analysis    49
4 Cultures of NSCs on PDMS    51
5 Movement Verification of Stretching Platform    55
5.1 Verification of Displacement    55
5.2 Verification of Strain    59
6 Stretching Experiments    60
6.1 Characterization via Optical Microscope    60
6.2 Identification of Neurite Outgrowth    64
6.3 Statistical Results    66
7 Immunocytochemistry    70
Chapter 5 Discussions    76
1 Characterization of Master and PDMS Substrate    76
2 Cytotoxicity Inspection of PDMS    77
3 Cultures of NSCs on PDMS    78
4 Movement Verification of Stretching Platform    79
5 Stretching Experiments    80
6 Immunocytochemistry    82

Chapter 6 Conclusions    84
References    85

                                

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