臺灣博碩士論文加值系統

大多數的細胞都需要基底以利其進行貼附、移動、生長、分化等行為。細胞在進行這些行為時，會受到基底的軟硬度或表面形態所影響。而細胞移動的速率對癌細胞的移轉是很重要的因素。本研究探討使用軟硬程度不同的基材搭配上不同表面特定形態，對於細胞貼附之後的移動能力之影響。
本研究使用人類黑色素腫瘤細胞 (Human melanoma)及人類黑色素細胞(human epidermal melanocytes)，以10：1及20：1兩種比例混合的矽酮(Silicon elastomer)及熟化劑矽酮樹脂溶液 (Silicon elastomer)調配出不同軟硬度的聚二甲基矽氧烷 (PDMS)為基底材料，並以材料試驗機 (MTS)量測PDMS的揚氏模數，使用微機電製程 (MEMS)技術翻模出不同的基材表面型態，包含平頂圓錐及長橫溝兩種，分別具有兩種不同線寬為1 μm及6 μm，與玻璃基材相對照。細胞於不同基材表面型態穩固貼附12小時後，連續觀察細胞位置6小時，使用Image Pro. Plus軟體做移動速率及方向分析計算。
以10：1及20：1兩種比例調配的PDMS其揚氏模數分別為3.04 ± 0.26及 1.44 ± 0.13 MPa，兩種細胞均會沿著與長橫溝平行的方向移動，且隨著線寬的增加而增加，在平頂圓柱及平的表面上則會無規則的排列與移動。人類黑色素腫瘤細胞隨長橫溝平行移動的比率，隨著線寬的減小而下降。人類黑色素腫瘤細胞貼附面積受平頂圓柱影響最為顯著，但人類黑色素細胞貼附面積卻不受基材軟硬度及表面形態所影響。
在所有基材上，人類黑色素細胞移動的速度均比人類黑色素腫瘤細胞移動的快。兩種細胞在玻璃基材上移動的最慢，在不同表面形態上，細胞在具有較軟的PDMS上移動的比較硬的PDMS快，且人類黑色素腫瘤細胞在較軟的PDMS所翻模出的6 μm平頂圓柱上移動的最快 (0.91 ± 0.06 μm/min)，人類黑色素細胞則在較軟的PDMS所翻模出的1 μm長橫溝上移動的最快 (1.02 ± 0.11 μm/min)。
綜合所得的結果，此研究證實人類黑色素腫瘤細胞比人類黑色素細胞對基材軟硬度及表面形態是較為敏感的。

Most cells are anchorage dependent. They require a surface to attach in order to migrate, proliferate, and differentiate. It is known several chemical factors can dramatically affect those functions of cells. Nevertheless, physical factors like stiffness and surface morphology of the substrata are also thought to influence cell behaviors. The purpose of this study was to measure the cell migration under different stiffness and structure of the substrata.
Human melanoma cells (A2058) and human epidermal melanocytes (HEMn) were used for this study. The polydimethylsiloxane (PDMS) with ratios of silicon elastomer/curing agent concentration at 10:1 and 20:1 were fabricated for soft substrata. The Young’s modulus of PDMS substrata with different ratios were measured by material testing system (MTS). PDMS samples were made into five forms, including flat, 6 μm cones, 6 μm grooves, 1 μm cones and 1 μm grooves by standard micro-electro-mechanical-systems (MEMS). Glass dish was used as control substrate. After cells were seeded on glass, flat PDMS, flat-top cone, or long groove PDMS for 12 hours to establish stable attachment, the cell images were taken by optical microscopy every 5 minutes for 6 hours. The speed and direction of cell migration were calculated by the coordinate of each cell on the images.
The modulus for PDMS with a ratio of 10:1 and 20:1 measured by MTS were 3.04 ± 0.26 and 1.44 ± 0.13 MPa respectively. For both cell types, parallel migration along the pattern occurred on the groove surface and the directional migration increased with pattern line width; however, both cell types did not show any preferred migration orientation n the cones and flat substrata. The frequency of A2058 cells moving along the groove decreased with smaller line width. Flat-top cone was the best surface morphology to influence A2058 cell area. In contrast, the cell area of HEMn cells appeared uniform to variation in surface morphology and substrate rigidity.
The speeds of HEMn cells were almost faster on all substrata when compared to the speed of A2058 cells. The migration speeds of both cell types on glass were the lowest and significantly faster on soft PDMS when compared to the stiff PDMS for most types of patterning substrate. The highest speeds of A2058 and HEMn cells were 0.91 ± 0.06 and 1.02 ± 0.11 μm/min was obtained on the soft 6 μm cones and soft 1 μm grooves respectively.
In conclusion, this study demonstrated that A2058 cells were more sensitive to the changes of surface morphology and substrate rigidity than HEMn cells.

ABSTRACT I
摘要 III
致謝 V
TABLE OF CONTENTS VI
LIST OF FIGURES IX
LIST OF TABLES XII
CHAPTER 1 INTRODUCTION 1
1.1 CELL MIGRATION 1
1.1.1 Migration process 2
1.1.2 The mechanism of cell migration 2
1.2 LITHOGRAPHY 5
1.3 POLYDIMETHLSILOXANE (PDMS) 6
1.4 THE VARIOUS FACTORS INFLUENCE CELL BEHAVIOR 7
1.4.1 Substrate rigidity 8
1.4.2 Surface morphology 10
1.4.3 The variance of different cell types 12
1.5 EXPERIMENTAL HYPOTHESIS 13
1.6 PURPOSE 14
1.7 FLOWCHART OF EXPERIMENT 15
CHAPTER 2 METHODOLOGY 18
2.1 MATERIALS AND INSTRUMENTS 18
2.2 CELL CULTURE 19
2.3 FABRICATION OF SILICON MOLD 20
2.4 FABRICATION OF MICROSTRUCTURED PDMS SUBSTRATES 21
2.5 MEASUREMENT OF YOUNG’S MODULUS OF PDMS 24
2.6 WATER CONTACT ANGLE MEASUREMENT 25
2.7 SEM/EDS ANALYSIS 26
2.8 TIME-LAPSE MICROSCOPY 26
2.9 ACCURACY OF THE SURFACE TOPOGRAPHY 28
2.10 EXAMINATION FOR CELL MORPHOLOGY 28
2.11 IMMUNOSTAING FOR ACTIN AND VINCULIN 28
2.12 IMAGE ANALYSIS AND STATISTICS 29
CHAPTER 3 RESULTS 30
3.1 PDMS MODULUS 30
3.2 SURFACE CHARACTERIZATION 31
3.3 SEM COMBINED WITH EDS ANALYSIS OF THE COLLAGEN COATING 32
3.4 ACCURACY OF SURFACE MORPHOLOGY 34
3.5 CELL MORPHOLOGY 36
3.6 CELL SPREADING AREA 40
3.7 ORGANIZATION AND ALIGNMENT OF CYTOSKELETON AND FOCAL ADHESIONS (FAS) 44
3.8 MIGRATION DIRECTION 49
3.9 MIGRATION SPEED 52
CHAPTER 4 DISCUSSION 58
4.1 CELL ALIGNMENT 58
4.1.1 Effect of line width on cell alignment 58
4.1.2 Effect of line depth on cell alignment 59
4.2 CELL SPREADING AREA 59
4.3 CELL MIGRATION ANGLE 60
4.3.1 Effect of line width on cell migration direction 60
4.3.2 Cell shape may be a key factor for cell migration direction 61
4.3.3 Effect of line depth on cell migration direction 61
4.4 CELL MIGRATION SPEED 62
4.4.1 Effect of substrate rigidity on cell speed 62
4.4.2 Effect of the time cell seeded on the substrate 62
4.4.3 Effect of cell area on cell speed 63
4.4.4 Effect of line width combine with cell shape on cell speed 64
4.4.5 Effect of different traction force in both cell types on cell speed 65
4.5 EXPERIMENTAL LIMITATIONS 67
4.6 FUTURE PROSPECTS 67
CHAPTER 5 CONCLUSIONS 68
REFERENCES 70
CURRICULUM VITAE 74

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