微流道應用於血管再生之研究｜國立清華大學博碩士論文庫

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研究生：	戈凱揚 Villalobos Gorday, Kaiser Alejandro
論文名稱：	微流道應用於血管再生之研究 The Study on Microchannels for Vasculature Regeneration
指導教授：	王潔 Wang, Jane
口試委員:	謝明佑鄭逸琳
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	110
中文關鍵詞：	微流道、Murray定律、人類臍帶内皮細胞、組織工程、PGSA
外文關鍵詞：	Microfluidic devices, Murray’s law, HUVEC, Tissue engineering, PGSA
相關次數：	點閱：4 下載：0
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器官再生被視為在組織工程中最具野心的挑戰之一，各種不同細胞錯綜複雜的三維排列，以及缺乏既有血管易導致組織壞死的特性，使其大大受限於臨床上的應用。因此，本研究致力於將Murray定律應用在建立帶有分枝狀設計之生物可分解微流道裝置，以冀能進一步用於血管之模擬。

在細胞實驗中，就材料的選擇而言，PGSA之丙烯酸化程度達到0.69或以上時，具有適於細胞外間質蛋白質貼附的接觸角，且呈現較高的細胞生長速率，此二特性被認為有助於細胞培養的使用；細胞外間質蛋白質的篩選中，混合纖連蛋白、第一型膠原蛋白與明膠的組合最利於細胞貼附，因此在後續的細胞培養中被選擇作為材料表面的塗層。不論是以相位差顯微鏡或是掃描式電子顯微鏡觀察，細胞都呈現高密度以及健康的型態。在建立微流道裝置方面，使用了電腦輔助設計和軟體來模擬應用Murray定律下，擁有最佳流體力學之情形，包含均勻的剪力分布與流動中最小的擾動。

使用DLP基層製造技術設計的微流道系統被證實有極高的應用性及快速成形特性，應用在培養細胞時，人臍靜脈內皮細胞大量出現在微流道的入口及出口處，並呈現健康的型態；以PGSA製造的微流道裝置用於培養內皮細胞時的良好結果，證實其對設計及製造具有網狀血管結構的器官助益良多。

關鍵字：微流道裝置、Murray定律、人臍靜脈內皮細胞、組織工程、PGSA

Solid organ regeneration is one of the most ambitions challenges in tissue engineering. The complex three-dimensional arrange of different kinds of cells and lack of preexisting vasculature leads to premature necrosis and limits its application in clinical uses. In this project, biodegradable microfluidic devices with bifurcating channels based on Murray’s law were fabricated.

PGSA DA:0.69 and higher showed preferable characteristics for cell seeding, with a suitable contact angle for ECM protein adhesion and high cell proliferation rate. A triple coating of fibronectin, type I collagen, and gelatin exhibited the highest cell adhesion rate and was selected as the coating recipe for subsequent cell seedings. Observations under phase contrast microscope and scanning electron microscope showed cells with healthy morphology and high cell density. Computer-assisted design and simulation software was applied for the design and simulation of microchannels. Designs based on Murray’s law exhibited superior fluid dynamics, with uniform shear stress distribution and minor fluctuations in flow speed.

DLP-AM fabricated microfluidic devices were robust with no signs of obstruction and the fabrication method proven to be practical for fast and precise prototyping. HUVEC seeded on microchannels showed healthy morphology and high cell number at the entrance and exit of the scaffold. Endothelial cell seeding in PGSA microfluidic devices was proven feasible and may contribute to the fabrication of solid organs with an embedded vascular network in the future.

Keywords: Microfluidic devices, Murray’s law, HUVEC, Tissue engineering, PGSA

Chapter 1: Introduction to Tissue Engineering and Vascularization    8
1 Tissue Engineering and Regenerative Medicine    8
1.1 Regeneration of Full Organs    8
1.2 Triad of Tissue Engineering    11
1.3 Scaffolds for Tissue Engineering    12
1.4 Criteria for Selection of Materials for Scaffold Fabrication    13
1.5 Common Choices of Materials for Tissue Engineering    17
2 Introduction to Biocompatible and Biodegradable Materials    20
2.1 Introduction to Polydimethylsiloxane (PDMS)    20
2.2 Introduction to Polyethylene (glycol) Diacrylate (PEGDA)    23
2.3 Introduction to Poly(glycerol sebacate) (PGS)    24
2.4 Introduction to Poly(glycerol sebacate) Acrylate (PGSA)    27
3 Introduction the Design of Microfluidic Devices    31
4 The Relationship between Vasculature Physiology and Murray’s Law    34
4.1 Derivation of Murray’s Law    35
4.2 Application of Murray’s Law    37
5    Motivation and Purpose    39
Chapter 2: Experimental Design and Methods    40
1 Materials and Reagents    40
2 Experimental Procedure    43
3 Material Synthesis    43
3.1 PDMS    43
3.2 PGS    43
3.3 PGSA    44
4 Fabrication of PEGDA and PGSA Films    45
5 Cell Culture    45
5.1 Cell Culture    46
5.2 Cell Subculture    46
5.3 Preparation of the Films    47
5.4 Cell Seeding    47
5.5 Contact Angle Assay    48
5.6 Resazurin Reduction Assay    48
5.7 Multisubstrate Coating Assay    49
5.8 Cell Survival Assay    50
5.9 Immunofluorescence Microscopy    51
5.10 Scanning Electron Microscopy    51
6 Computer-Assisted Design and Simulation (CAD/CAS) of Microfluidic Devices (CAD/CAS)    52
6.1 Design of Microchannels    52
6.2 Reynolds Number and Entrance Length    53
6.3 Computer-Assisted Drawing and Simulation of Microfluidic Devices    54
7 Fabrication of Microfluidic Devices    55
7.1 Fabrication of Casting Molds    55
7.2 Printing Mold    55
7.3 3D Printing via DLP-AM System    56
7.4 Characterization of Microchannels    56
7.5 Assembly of Devices    57
7.6 Ink Perfusion Tests    59
8 Cell Seeding in Microchannels    60
9 Statistical Analysis    61
Chapter 3: Results and Discussion    62
1 Material Selection through Contact Angle and Metabolic Activity    62
1.1 Water Contact Angle Assay of Different Materials    62
1.2 Cell Viability Assay through Resazurin Reduction Test    65
1.3 Multisubstrate Coating Assay    68
1.4 Cell Survival Assay    72
2 Flow Simulation Analysis    77
3 Characterization of Microchannels    81
3.1 Ink Perfusion Test    81
3.2 Scanning Electron Microscope Imaging of Microchannels    83
4 Observation of HUVEC on Scaffold under Phase Contrast and Scanning Electron Microscope    86
5 Cell Seeding Inside Microchannel    89
Chapter 4: Conclusion    93
Chapter 5: Future Work    95
1 Bioreactor to Optimize Cell Seeding Inside Microchannels    95
2 Characterization of Cells Inside Microchannels    95
3 Gene Expressions of Cells Inside Microchannels    95
4 3D Printing of Microchannels Inside Scaffolds for Liver Scaffold    95
Appendix    97
A. Yellow pigmentation in PGSA pre-polymer.    97
B: Cell Counting by Immunofluorescence Microscopy.    98
Chapter 6: References    99
                                

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