利用改質氧化矽奈米結構來探討細胞結構之反應

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研究生：	楊中堯 Yang, Chung-Yao
論文名稱：	利用改質氧化矽奈米結構來探討細胞結構之反應 Probing Cellular Structural Responses through Functionalized Oxidized Silicon Nanosponges
指導教授：	葉哲良 Yeh, J. Andrew
口試委員:	沈湯龍陳文翔陳致真鄭兆珉饒達仁周涵怡
學位類別：	博士 Doctor
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：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	151
中文關鍵詞：	氧化矽、表面改質、細胞
相關次數：	點閱：3 下載：0
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This paper describes the micropatterning of mammalian cells onto silicon surfaces with nanometer-scale regular features made through a nanofabrication approach. The ability to modulate cell–substrate interactions has provided a number of exciting insights in cell-relevant research including stem cell differentiation, cell survival, mechanotaxis, and biomaterials. The propose of this study is twofold; (i) a nanofabrication method, which can be used to create nanometer-scale regular features on silicon surfaces with the surface characteristics of hydrophobicity, has been developed through a combination of the conventional photolithography approach and the chemical-based surface modification, enabling us to probe the cell-substrate interactions; and (ii) the micropatterning of a variety of mammalian cells (Chinese hamster ovary and HIG-82 in this study) on silicon surfaces has been demonstrated. The cytoskeletal and morphologic changes in living cells attached to our nanopatterns on silicon surfaces have been examined as well via fluorescence microscopy. This study, we believe, would provide us a better understanding of the cell-substrate interactions, and have a wide range of applications such as the development of biocompatible nanofabrication methods, biomaterials, functional biomedical devices, as well as tissue engineering.

本篇論文利用改質後的氧化矽奈米海綿結構來探討其對細胞結構之反應，本文所提出的研究有兩個重點，一為製作奈米結構的流程方法，另一為利用此奈米結構來控制細胞的生長。本方法結合物理性的奈米結構以及化學性的表面鍍膜來探討其對於細胞生物學的影響，此奈米結構是以化學蝕刻所製作出來，而表面鍍膜則是利用分子氣相沉積來做改質。不同於物理性製作奈米結構之方法，利用化學蝕刻可以達到晶圓等級的範圍，而分子氣相沉積改質也與以往利用自組裝分子來做改質不相同，不僅大大縮短反應時間，其分子膜的品質也非常良好，不會因為液體自組裝分子的大小而影響到奈米結構的形貌。除此之外，此製作流程也極易整合於目前半導體製程，來達到大量製作實驗樣品的目的。利用此製程所做出之奈米結構將可以更深入探討細胞生長於三維奈米結構上之行為，本文使用兩種不同的細胞做研究，一為中國倉鼠卵巢細胞(CHO)，另一為兔滑液膜細胞(HIG-82)，利用此奈米結構並搭配不同表面官能基來探討細胞之結構形貌，並利用此結構成功的引導細胞的生長。此研究將可以使學者更容易了解細胞生物學的一些反應，以及細胞與材料基板之間的交互作用，更可將其應用於組織工程，來達到體外大量培養有方向性的組織細胞，相信本研究可以為生物學家帶來更多的衝擊

ACKNOWLEDGEMENT    I
ABSTRACT    II
中文摘要    III
CONTENTS    IV
CAPTIONS OF FIGURES    VIII
TABLES    XIV

CHAPTER I: INTRODUCTION    1
1.1 BACKGROUND    1
1.2 MICRO- AND NANOENGINEERING CONTROL OF CELLULAR ENVIRONMENT    3
1.2.1 CELL-SUBSTRATE INTERACTIONS    3
1.2.2 CELL SHAPE    6
1.2.3 CELL ADHESION    8
1.2.4 THREE-DIMENSIONAL SCAFFOLDS    10
1.2.5 CELL-CELL INTERACTIONS    12
1.2.6 CELL-SOLUBLE FACTOR INTERACTIONS    13
1.3 NANOENGINEERING SURFACE FOR CELL CULTURE    14
1.4 OBJECTIVES    27
CHAPTER II: MATERIALS AND METHODS FOR TISSUE ENGINEERING    29
2.1 MATERIALS FOR THREE-DIMENSIONAL SCAFFOLD    29
2.1.1 POLYMERS    31
2.1.2 BIODEGRADABLE SCAFFOLDS    32
2.2 METHODS FOR GENERATING SCAFFOLDS    33
2.2.1 3D PRINTING    35
2.2.2 PHOTOLITHOGRAPHY    36
2.2.3 SOFT LITHOGRAPHY    39
2.2.3.1. MICROCONTACT PRINTING    40
2.2.3.2 MICROFLUIDIC PATTERNING    41
2.2.3.3. STENCIL PATTERNING    42
2.2.4 ELECTROSPINNING    43
2.2.5 LAYER-BY-LAYER DEPOSITION    45
2.2.6 NANOTOPOGRAPHY GENERATION    47
CHAPTER III: CELL MORPHOLOGY AND ADHESION ON FUNCTIONALIZED SILICON SURFACES    49
3.1 NANO-TOPOGRAPHIC OXIDIZED SILICON SURFACES FOR CELL CULTURE    49
3.2 SURFACE CHARACTERISTICS OF SUBSTRATES    52
3.2.1 SURFACE TREATMENT OF OXIDIZED SILICON SUBSTRATES AND NANOSPONGES    52
3.2.2 SURFACE HYDROPHOBICITY AND TOPOLOGY    54
3.3 CHO CELLS ON DIFFERENT SUBSTRATES    59
3.3.1 CELL CULTURE    59
3.3.2 CELL MORPHOLOGIES    60
3.3.2.1 PRE-PROCEDURE FOR SEM IMAGING    60
3.3.2.2 SEM IMAGES OF CELLS ON DIFFERENT SURFACES    61
3.3.3 CELL CYTOSKELETON ON DIFFERENT SUBSTRATES    65
3.3.3.1 PRE-PROCEDURE FOR LASER SCANNING CONFOCAL MICROSCOPY IMAGING    65
3.3.3.2 LSCM IMAGES OF CELLS ON DIFFERENT SURFACES    65
3.3.4 CELL ADHESION AND VIABILITY ON DIFFERENT SUBSTRATES OVER TIME    69
3.3.4.1 CELL ADHESION ASSAY    69
3.3.4.2 MTT ASSAY    69
3.3.4.3 CELL ADHESION AND VIABILITY    70
3.3.5 BIOCHEMICAL PROPERTIES    72
3.3.5.1 WESTERN BLOTTING    73
3.3.5.2 BIOCHEMICAL INSIGHTS    73
3.5 SUMMARY    82
CHAPTER IV: MICROPATTERNING OF MAMMALIAN CELLS ON FUNCTIONALIZED SILICON SURFACES    84
4.1 INTRODUCTION OF CELL MICROPATTERNING    84
4.2 FABRICATION OF PATTERNED OXIDIZED SILICON NANOSPONGES    88
4.3 SURFACE CHARACTERISTICS OF SILICON NANOSPONGES    90
4.4 CHO AND HIG-82 CELLS ON MICROPATTERNED SILICON NANOSPONGES    94
4.4.1 CELL CULTURE    94
4.4.2 CELL MORPHOLOGIES    95
4.4.2.1 PRE-PROCEDURE FOR SEM IMAGING    95
4.4.2.2 SEM IMAGES OF CELLS ON DIFFERENT SURFACES    96
4.4.3 CELL CYTOSKELETON ON DIFFERENT SUBSTRATES    101
4.4.3.1 PRE-PROCEDURE FOR EPI-FLUORESCENT MICROSCOPY IMAGING    101
4.4.3.2 EPI-FLUORESCENT IMAGES OF CELLS ON SILICON NANOSPONGES    101
4.5 SUMMARY    104
CHAPTER V: CONCLUSION    106
CHAPTER VI: FUTURE PERSPECTIVES    109
6.1 CELLULAR ANALYSES    109
6.2 CELL-BASED SENSING    111
6.3 TISSUE ENGINEERING    113
REFERENCES    116
APPENDIX A    127
A.1 MOLECULAR VAPOR DEPOSITION    127
A.1.1 PRINCIPLES    128
APPENDIX B    131
B.1 ATOMIC FORCE MICROSCOPE (AFM)    131
B.1.1 PRINCIPLES OF TAPPING-MODE AFM    134
B.1.2 BEAM DEFLECTION DETECTION    136
B.1.3 AFM CANTILEVERS    136
B.1.4 FORCE CURVE MEASUREMENT    139
APPENDIX C    142
C.1 NANOCHIP FOR CELL CULTURE -- PROCESS FLOW    142
CURRICULUM VITAE    149

                                

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