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研究生:曾鈞澤
研究生(外文):Tseng, Chun-Tse
論文名稱:3T3-L1脂肪前驅細胞分化及分選之微流體晶片發展
論文名稱(外文):Development of a microfluidic device for 3T3-L1 pre-adipocyte differentiation and sorting
指導教授:許佳賢許佳賢引用關係
指導教授(外文):Hsu, Chia-Hsien
口試委員:洪明秀吳嘉哲
口試委員(外文):Hung, Ming-ShiuWu, Chia-Che
口試日期:2023-07-27
學位類別:碩士
校院名稱:國立清華大學
系所名稱:奈米工程與微系統研究所
學門:工程學門
學類:材料工程學類
論文種類:學術論文
論文出版年:2023
畢業學年度:111
語文別:英文
論文頁數:48
中文關鍵詞:微流體晶片聚苯乙烯塊材之熱接合二維脂肪細胞模型膠原酶流體剪應力
外文關鍵詞:Microfluidic chipThermal bonding of polystyrene (PS)2D adipocytes modelCollagenaseFluid shear stress
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肥胖症的流行儼然已成為全世界現今所共同面對的挑戰之一,根據世界衛生組織的統計數據,每年有280萬人因肥胖相關症狀而導致死亡。因此,應對這一健康問題,製藥公司積極地研發抗肥胖藥物。然而,在眾多的體外3T3-L1脂肪細胞的研究中,3T3-L1脂肪前驅細胞的分化,細胞的異質性是一個容易造成實驗誤差的問題。因此,開發一種可以廣泛應用在肥胖症狀研究的微流體裝置,來解決這個問題,對於增進在導致肥胖相關機制的了解是相當重要的。在本研究中,通過精確控制膠原酶作用在成熟脂肪細胞的反應時間和調控不同的流體流速,我們成功地分選到單一顆分化或成熟的脂肪細胞。此外,為了製作能夠用來長時間使用的微流體晶片,我們開發了一種利用自製夾具來達成聚苯乙烯(PS)材質的微流體晶片的熱接合方法。總體來說,本研究展示了一種能夠長期使用的微流體晶片,且此晶片能夠進行3T3-L1前驅脂肪細胞的培養和分化,以及能夠分選出成熟或分化的脂肪細胞。
The widespread obesity epidemic presents one of the most significant challenges to public health. World Health Organization statistics 2.8 million people die due to being overweight or obese each year. Consequently, in response to this health issue, pharmaceutical companies are actively working on developing anti-obesity medications. However, among the extensive research of in vitro 3T3-L1 adipocyte cell models, cell heterogeneity during 3T3-L1 pre-adipocyte differentiation is a concerned issue leading to experimental error. Therefore, the development of a widely-used microfluidic platform for obesity research to solve this problem is crucial to advance our understanding of obesity-related mechanisms. In this study, by precisely controlling the collagenase treatment time and fluid flow rate, we successfully sorted the individual differentiated adipocytes. Moreover, to fabricate the microfluidic chip, an assembly method for the Polystyrene (PS) microfluidic chip is developed, utilizing thermal bonding with a customized clamp. In sum, this research presents the development of a long-term used microfluidic chip that integrates the culturing and differentiation of 3T3-L1 pre-adipocytes, along with the sorting of the differentiated adipocytes.
摘要 2
ABSTRACT 3
Table of contents i
List of illustrations iii
Chapter 1. Introduction 1
1.1 Obesity 1
1.2 Adipose tissue and adipocytes morphology 2
1.3 In vitro culture of adipocyte cell and culture techniques 4
1.3.1 Adipocyte cell models 4
1.3.2 Cell heterogeneity in 3T3-L1 pre-adipocytes differentiation 6
1.3.3 Current methods for sorting adipocytes 8
1.4 Cell dissociation 8
1.5 Application of the shear stress in microfluidic device 10
1.6 Thermal bonding of thermoplastic materials 11
Chapter 2. Research Objectives 14
Chapter 3. Materials and Methods 16
3.1 Chip fabrication and experimental protocol 16
3.1.1 Setup of cell sorting experiment 17
3.2 Thermal bonding procedure 19
3.2.1 Operation of customized double plate clamp 20
3.3 Bonding quality of microfluidic chip 22
3.3.1 Bonding strength test via applying tensile test experiment 22
3.3.2 Observing cell leakage phenomenon within the microfluidic chip
23
3.4 3T3-L1 cell culturing and differentiating into adipocytes 23
3.5 Methodology for identifying cell diameter and cell cluster projective area in sorting experiments 24
3.6 Statistical Analyses 25
Chapter 4. Experimental results and Discussion 27
4.1 Chip design and operation 27
4.2 Evaluation of PS-PS bonding quality 28
4.2.1 Optimization between bonding strength and minimum channel deformation 28
4.2.2 Optimal assistive tool for chip assembling: Customized clamp vs. Toggle clamp vs. 3M double tape 31
4.2.3 Observing leakage of the microfluidic chip with adipocytes 33
4.3 Cell sorting of differentiated adipocytes 35
4.3.1 Effects of varying collagenase treatment time on sorting differentiated adipocytes 35
4.3.2 Influence of flow rate on sorting differentiated adipocytes 38
Chapter 5. Conclusion 42
Chapter 6. References 43
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