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研究生:王啟睿
研究生(外文):Chi-Jui Wang
論文名稱:微流碟盤系統應用於血液中外吐小體之免疫親合抓取之研究
論文名稱(外文):Centrifugal microfluidic platform enabling immunoaffinity-based exosome enrichment from whole blood
指導教授:胡文聰胡文聰引用關係
口試日期:2017-07-26
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:應用力學研究所
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:36
中文關鍵詞:微流碟盤外吐小體乳癌自動化免疫親合抓取
外文關鍵詞:microfluidicexosomesbreast cancerautomatedimmunomagnetic approach
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癌症轉移是目前最主要的癌症死亡原因,對於其轉移的機制至今仍待釐清。透過分析現今所認定之癌症轉移因子,盼望對於其轉移的預防能有更進一步的貢獻。近年來,外吐小體已被許多研究證實在癌症轉移中扮演著相當重要的角色,而用其當作早期癌症轉移的標的也漸趨明朗。如何精準且快速的抓取與分析高純度的外吐小體也就成為現今很重要的一個課題。本研究提出一套能運用在自動化機台的微流碟盤系統。此系統由微結構碟盤與特殊設計的微量離心管承載器所構成,碟盤可直接從全血開始進行自動化血漿分離以及外吐小體的磁珠免疫親合抓取,其免疫磁珠與血漿比例和外吐小體抓取效率則是經由100到600微升的全血進行驗證,而結果是由西方墨點法進行蛋白檢測。此外,透過與超高速離心法和高分子試劑抓取的比較,實驗結果證實,碟盤系統可從三位乳癌患者成功的分離出血漿中的外吐小體。且可從全血分離98.3%的紅血球以達到血漿分離。此系統透過自動化機台和免疫親合法的專一抗體結合抗原特性,減少了人為的操作實驗的誤差,同時省去了超高速離心繁瑣步驟和常用試劑的非專一抓取的問題。盼望此系統能對於未來臨床研究者提供妥善的協助。
Cancer management can be better served by suitable biomarkers ranging from diagnosis and monitoring of therapeutic progress. Over the past few years, clinical relevance of exosomes as a marker during tumor progression and early disease detection has been validated. However, the enrichment of exosomes remains technically challenging, where purity, reproducibility and automation are highly desirable. In this thesis, a centrifugal microfluidic platform was presented to enrich exosomes directly from blood. The platform contains a microfluidic disk and a mechanism to collect plasma into an Eppendorf tube. A range of parameters of immunomagnetic beads to plasma ratio and system performance could be obtained from 100 to 600 μl of human whole blood. Western blotting was used for protein quantification. Besides, the performance of exosome enrichment was compared with that from ultracentrifugation and a commercial exosome isolation kit. Results showed that the microfluidic device successfully enriches exosomes from three breast cancer patients directly from whole blood. Averaged 98.3% red blood cells from whole blood was depleted in the plasma separation process. Taken together, our microfluidic platform provides a simple-to-use and robust approach to enrich specific exosomes by recognizing the exosomal surface markers. Moreover, the automated system reduces variation in operator biases and may serve as a standard device for clinical uses.
口試委員會審定書 i
致謝 ii
中文摘要 iii
Abstract iv
目錄Table of Contents vi
圖目錄 List of figures viii
表目錄 List of tables ix
Chapter 1. Introduction 1
1.1 Clinical relevance of exosome 1
1.2 Technologies for exosomes enrichment 2
1.3 Development of exosomes enrichment via microfluidic disk system 6
Chapter 2. Design Feature and Methodology 7
2.1 Plasma separation microchannel network 7
2.2 Eppendorf design for exosome enrichment 11
2.3 System setup 12
Chapter 3. Materials 13
3.1 Materials 13
3.1.1 Disk and Eppendorf holder fabrication 13
3.1.2 Preparation of plasma 14
3.1.3 Preparation of conditioned medium 14
3.1.4 Reagents 15
Chapter 4. Methods 16
4.1 Exosomes enrichment 16
4.1.1 Ultra-centrifugation 16
4.1.2 Immunomagnetic approach 18
4.1.3 Total exosome isolation precipitation 18
4.1.4 Centrifugal microfluidic platform 18
4.2 Exosome detection 19
4.2.1 Electron microscopy 19
4.2.2 Immunoblotting 20
Chapter 5. Results and Discussion 21
5.1 Overview of immunoaffinity-based exosome enrichment via disk platform 21
5.2 Performance of plasma separation from whole blood using the disk platform 22
5.3 Immunomagnetic exosome enrichment: disk-based versus tube-based approached 25
5.4 Comparison of performance among disk system, total exosome isolation kit, and ultracentrifugation 29
Chapter 6. Concluding remarks 32
References 34
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3. Pantel, K., R.H. Brakenhoff, and B. Brandt, Detection, clinical relevance and specific biological properties of disseminating tumour cells. Nat Rev Cancer, 2008.8(5): p. 329-340.
4. Alix-Panabières, C. and K. Pantel, Circulating Tumor Cells: Liquid Biopsy of Cancer. Clinical Chemistry, 2013. 59(1): p. 110-118.
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8. Costa-Silva, B., et al., Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol, 2015. 17(6): p. 816-826.
9. Becker, A., et al., Extracellular Vesicles in Cancer: Cell-to-Cell Mediators of Metastasis. Cancer Cell. 30(6): p. 836-848.
10. Witwer, K.W., et al., Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. Journal of Extracellular Vesicles, 2013.2(1): p. 20360.
11. Chiou, N.-T. and K.M. Ansel, Improved exosome isolation by sucrose gradient fractionation of ultracentrifuged crude exosome pellets. 2016.
12. Baranyai, T., et al., Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods. Plos One, 2015. 10(12).
13. Gámez-Valero, A., et al., Size-Exclusion Chromatography-based isolation minimally alters Extracellular Vesicles’ characteristics compared to precipitating agents. 2016. 6: p. 33641.
14. Sodar, B.W., et al., Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection. Sci Rep, 2016. 6: p.24316.
15. Akagi, T., et al., On-Chip Immunoelectrophoresis of Extracellular Vesicles Released from Human Breast Cancer Cells. PLoS ONE, 2015. 10(4): p. e0123603.
16. Wang, Z., et al., Ciliated micropillars for the microfluidic-based isolation of nanoscale lipid vesicles. Lab on a chip, 2013. 13(15): p. 2879-2882.
17. Van Deun, J., et al., The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. Journal of Extracellular Vesicles, 2014.3(1): p. 24858.
18. Rider, M.A., S.N. Hurwitz, and D.G. Meckes Jr, ExtraPEG: A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles. 2016. 6: p. 23978.
19. Brownlee, Z., et al., A novel “salting-out” procedure for the isolation of tumorderived exosomes. Journal of immunological methods, 2014. 407: p. 120-126.
20. Kobavashi, M., et al. Development of microfluidic devices with polyethylene glycol-lipid-modified adsorption surface for high- Throughput isolation of
exosomes from human serum. in 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013. 2013.
21. Yoshioka, Y., et al., Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen. 2014. 5: p. 3591.
22. Ko, J., et al., Smartphone-enabled optofluidic exosome diagnostic for concussion recovery. Scientific Reports, 2016. 6.
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Extracellular Vesicles, 2013. 2(1): p. 20920.
24. Liga, A., et al., Exosome isolation: a microfluidic road-map. Lab on a Chip, 2015.15(11): p. 2388-2394.
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30. 徐韋凡, 負篩選卡匣式碟盤系統應用於血液中循環腫瘤細胞之分離抓取之研究. 1921.
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33. Kim, J., et al., Isolation of High-Purity Extracellular Vesicles by Extracting Proteins Using Aqueous Two-Phase System. PLOS ONE, 2015. 10(6): p. e0129760.
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