濃度梯度微晶片製作分析及細胞運動檢測應用

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姓名

周郁舜(Yu-shun Chou) 查詢紙本館藏

畢業系所

機械工程學系

論文名稱

濃度梯度微晶片製作分析及細胞運動檢測應用
(Microfluidic chemotaxis device for measuring the cell migration of the placenta-derived multipotent cells)

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摘要(中)

由於微流體晶片具有降低成本、提升分析效率與可進行即時觀察等優點，對於生醫科技來說擁有相當大的發展潛力，因此近年來許多學者將微流體裝置應用到生醫領域方面的研究。本文設計製作一個微流體裝置，讓人類胎盤源多功能幹細胞在其系統區域內進行正常的生長與運動，而且此微流體晶片可以產生類似線性分佈的濃度梯度。此外藉由實驗來觀察人類胎盤源多功能幹細胞於第一型膠原蛋白濃度梯度分佈下其運動之行為，且膠原蛋白濃度梯度的初始範圍為0-3μM，根據實驗結果顯示在注入流率為1 μL/min下，人類胎盤源多功能幹細胞無法進行正常的生長與運動，至於注入流率為0.05 μL/min時，人類胎盤源多功能幹細胞能夠正常的生長並進行遷移的行為，但是並沒有明顯的化學趨向性反應。

摘要(英)

Recently, microfluidic device has been applied to the biological and medical fields because of its huge potential for high throughput screening and advantages such as experiment cost down, increasing analysis efficiency and real time observation.This study aimed to create a microfluidic concentration generator for measuring the chemotactic migration of the placenta- derived multipotent cells (PDMCs) responding to type I collagen. The microfluidic device could generate linear-like concentration gradients by using cascade branches of micro-channels.
The collagen solution was pumped in concentration generator at two flow rates of 1 μL/min and 0.05 μL/min respectively to establish the type I collagen gradients. The type I collagen concentration gradient ranged between 0 and 3.03 μM. According to the experimental results, the PDMCs scarcely migrated and were washed off by the flow at 1 μL/min. The PDMCs showed random walks when the flow rate was 0.05 μL/min. However, the PDMCs showed no directional migration in response to the collagen gradient. This might be because the concentration of 3μM had saturated the chemotactic behavior of the cells.

關鍵字(中)

★ 微流體晶片
★ 人類胎盤源多功能幹細胞
★ 第一型膠原蛋白
★ 濃度梯度分佈
★ 化學趨向性

關鍵字(英)

★ microfluidic device
★ placenta-derived multipotent cell
★ type I collagen
★ concentration gradient
★ chemotaxis

論文目次

摘要 ............................................................................................................................ I
Abstract ................................................................................................................... II
致謝 ......................................................................................................................... III
目錄 ......................................................................................................................... IV
表目錄 ..................................................................................................................... VI
圖目錄 .................................................................................................................... VII
第一章緒論.............................................................................................................. 1
1.1 前言 ........................................................................................................ 1
1.2 微機電系統與微流體晶片 ...................................................................... 3
1.3 文獻回顧 ................................................................................................. 5
1.3.1 細胞運動 ...................................................................................... 5
1.3.2 微流體晶片進行化學趨向性實驗 ............................................... 7
1.4 研究目的 ................................................................................................. 9
第二章微晶片設計與製程 .....................................................................................13
2.1 製程設備 ................................................................................................13
2.2 材料選擇 ................................................................................................15
2.2.1 SU-8光阻劑 ...............................................................................15
2.2.2 聚二甲基矽氧烷（PDMS） .......................................................15
2.3 微影製程 ................................................................................................16
2.3.1 光罩設計與製作 .........................................................................17
2.3.2 晶圓片清潔（wafer cleaning） ..................................................19
2.3.3 光阻塗佈（spin coating） ..........................................................19
2.3.4 軟烤（soft bake） .......................................................................21
2.3.5 曝光（exposure） .......................................................................22
2.3.6 曝後烤（post exposure bake） ...................................................22
2.3.7 顯影（develop） ........................................................................23
2.3.8 硬烤（hard bake）......................................................................24
2.4 以PDMS製作微流體晶片 ....................................................................24
2.4.1 翻模過程 .....................................................................................25
2.4.2 脫模與連接器（Connector）製作..............................................26
2.4.3 氧電漿接合 .................................................................................26
2.5 微流體晶片之測詴方法與測詴結果 .....................................................28
2.5.1 藥品配製 .....................................................................................28
2.5.2 膠原蛋白濃度梯度之測詴方法 ..................................................30
2.5.3 濃度梯度之測詴結果 .................................................................31
2.5.4 FITC濃度梯度測詴 ....................................................................33
第三章理論模型與數値方法 ................................................................................48
3.1基本假設與物理系統 .............................................................................48
3.2統御方程式 ............................................................................................49
3.3邊界條件 ................................................................................................49
3.4參數設置 ................................................................................................51
3.4.1流體密度 ......................................................................................52
3.4.2流體黏滯係數 ..............................................................................52
3.4.3膠原蛋白之擴散係數 ..................................................................53
3.5數值方法 ................................................................................................54
3.5.1 COMSOL Multiphysics 簡介 .....................................................54
3.5.2網格設置 ......................................................................................55
3.6數值計算之結果 .....................................................................................55
3.6.1速度分佈之模擬分析 ..................................................................56
3.6.2濃度分佈之模擬分析 ..................................................................56
3.6.3 FITC擴散係數之擬合分析 ........................................................58
第四章實驗方法 .....................................................................................................74
4.1藥劑配製 ................................................................................................74
4.2細胞培養 ................................................................................................76
4.3實驗操作步驟 .........................................................................................79
4.3.1實驗前系統的準備 ......................................................................79
4.3.2細胞種植 ......................................................................................81
4.3.3以藥物建立濃度梯度與影像觀察 ...............................................82
第五章實驗結果與討論 .........................................................................................88
5.1細胞靜態培養 .........................................................................................88
5.2 PDMCS對第一型膠原蛋白濃度梯度之反應 ........................................89
5.3實驗結果討論 .........................................................................................90
第六章結論與未來展望 ....................................................................................... 100
6.1結論 ...................................................................................................... 100
6.2未來展望 .............................................................................................. 101
參考文獻 ................................................................................................................ 102

參考文獻

Ananthakrishnan, R., and Ehrlicher, A., 2007. The forces behind cell movement. International Journal of Biological Sciences 3(5), 303 - 317.
Becker, H., and Gärtner, C., 2008. Polymer microfabrication technologies for microfluidic systems. Analytical and Bioanalytical Chemistry 390(1), 89 - 111.
Behar, T.N., Schaffner, A.E., Colton, C.A., Somogyi, R., Olah, Z., Lehel, C., and Barker, J.L., 1994. GABA-induced chemokinesis and NGF-induced chemotaxis of embryonic spinal cord neurons. The Journal of Neuroscience 14(1), 29 - 38.
Brehm, W., Burk, J., Delling, U., Gittel, C., and Ribitsch, I., 2012. Stem cell-based tissue engineering in veterinary orthopaedics. Cell Tissue Res. 347, 677 - 688.
Brignier, A.C., and Gewirtz, A.M., 2010. Embryonic and adult stem cell therapy. Journal of Allergy and Clinical Immunology 125(2), 336 - 344.
Carter, S.B., 1965. Principles of Cell Motility: The Direction of Cell Movement and Cancer Invasion. Nature 208, 1183 - 1187.
Cimetta, E., Cannizzaro, C., James, R.,Biechele, T., Moon, R.T., Elvassore, N., and Vunjak-Novakovic, G., 2010. Microfluidic device generating stable concentration gradients for long term cell culture: application to Wnt3a regulation of β-catenin signaling. Lab on a Chip 10(23), 3277 - 3283.
Dertinger, S.K.W., Chiu, D.T., Jeon, N.L., and Whitesides, G.M., 2001. Generation of gradients having complex shapes using microfluidic networks. Analytical Chemistry 73(6), 1240 - 1246.
Faley, S.L., Copland, M., Wlodkowic, D., Kolch, W., Seale, K.T., Wikswo, J.P., and Cooper, J.M., 2009. Microfluidic single cell arrays to interrogate signalling dynamics of individual, patient- derived hematopoietic stem cells. Lab on a Chip 9, 2659 - 2664.
Figallo, E., Cannizzaro, C., Gerecht, S., Burdick, J.A., Langer, R., Elvassore, N., and Vunjak-Novakovic, G., 2007. Micro-bioreactor array for controlling cellular microenvironments. Lab on a Chip 7(6), 710 - 719.
Gelman, R.A., and Piez, K.A., 1980. Collagen fibril formation in vitro. The Journal of Biological chemistry 255(17), 8098 - 8102.
Jeon, N.L., Baskaran, H., Dertinger, S.K.W., Whitesides, G.M., Van de Water, L., and Toner, M., 2002. Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device. Nature Biotechnology 20(8), 826 - 830.
Jeon, N.L., Dertinger, S.K.W., Chiu, D.T., Choi, I.S., Stroock, A.D., and Whitesides, G.M., 2000. Generation of solution and surface gradients using microfluidic systems. Langmuir 16(22), 8311 - 8316.
Jeong, O.C., Park, S.W., Yang, S.S., and Pak, J.J., 2005. Fabrication of a peristaltic PDMS micropump. Sensors and Actuators A 123 - 124, 453 - 458.
Kim, S., Kim, H.J., and Jeon, N.L., 2010. Biological applications of microfluidic gradient devices. Integrative Biology 2(11-12), 584 - 603.
Langer, R., and Vacanti, J.P., 1993. Tissue Engineering. Science 26, 920 - 926.
Lauffenburger, D.A., and Horwitz, A.F., 1996. Cell migration: A physically integrated molecular process. Cell 84(3), 359 - 369.
Lee, D., and Chen Y.T., 2011. Mixing in tangentially crossing microchannels. American Institute of Chemical Engineers Journal 57(3), 571 - 580.
Lee, J. N., Jiang, X., Ryan, D., and Whitesides, G.M., 2004. Compatibility of mammalian cells on surfaces of poly(dimethylsiloxane). Langmuir 20(26), 11684 - 11691.
Lin, F., and Butcher, E.C., 2006. T cell chemotaxis in a simple microfluidic device. Lab on a Chip 6(11), 1462 - 1469.
Lin, F., Nguyen, C.M., Wang, S.J., Saadi, W., Gross, S.P., and Jeon N.L., 2004. Effective neutrophil chemotaxis is strongly influenced by mean IL-8 concentration. Biochemical and Biophysical Research Communications 319(2), 576 - 581.
Lin, F., Nguyen, C.M., Wang, S.J., Saadi, W., Gross, S.P., and Jeon N.L., 2005. Neutrophil migration in opposing chemoattractant gradients using microfluidic chemotaxis devices. Annals of Biomedical Engineering 33(4), 475 - 482.
Liu, Y., Sai, J., Richmond, A., and Wikswo, J.P., 2008. Microfluidic switching system for analyzing chemotaxis responses of wortmannin- inhibited HL-60 cells. Biomed Microdevices 10(4), 499 - 507.
McCarthy, J.B., Palm, S.L., and Furcht, L.T., 1983. Migration by haptotaxis of a schwann cell tumor line to the basement membrane glycoprotein laminin. The Journal of Cell Biology 97, 772 - 777.
Mengeaud, V., Josserand, J., and Girault, H.H., 2002. Mixing processes in a zigzag microchannel: Finite element simulations and optical study. Analytical Chemistry 74(16), 4279 - 4286.
Mosadegh, B., Saadi, W., Wang, S.J., and Jeon, N.L., 2008. Epidermal growth factor promotes breast cancer cell chemotaxis in CXCL12 gradients. Biotechnology and Bioengineering 100(6), 1205 - 1213.
Nguyen, T.T., Pham, M., and Goo, N.S., 2008. Development of a peristaltic micropump for bio-medical applications based on mini LIPCA. Journal of Bionic Engineering 5(2), 135 - 141.
Okada, S., Ishii, K., Yamane, J., Iwanami, A., Ikegami, T., Katoh, H., Iwamoto, Y., Nakamura, M., Miyoshi, H., Okano, H.J., Contag, C.H., Toyama, Y., and Okano, H., 2005. In vivo imaging of engrafted neural stem cells: its application in evaluating the optimal timing of transplantation for spinal cord injury. FASEB Journal 19(13), 1839 -1841.
Oster, G.F., Murray, J.D., and Harris, A.K., 1983. Mechanical aspects of mesenchymal morphogenesis. J. Embryol. exp. Morph. 78, 83 - 125.
Pamme, N., Eijkel, J.C.T., Manz, A., 2006. On-chip free-flow magnetophoresis: Separation and detection of mixtures of magnetic particles in continuous flow. Journal of Magnetism and Magnetic Materials 307(2), 237 - 244.
Randall, G.C., and Doyle, P.S., 2005. Permeation-driven flow in poly (dimethylsiloxane) microfluidic devices. Proceedings of the National Academy of Sciences of the United States of America 102(31), 10813 - 10818.
Saxena, A.K., 2005. Tissue engineering: Present concepts and strategies. Journal of Indian Association of Pediatric Surgeons 10(1), 14 - 19.
Tabata, Y., 2005. Significance of release technology in tissue engineering. Drug Discovery Today 10(23-24), 1639 - 1646.
Teixeira, A.I., Abrams, G.A., Bertics, P.J., Murphy, C.J., and Nealey, P.F., 2003. Epithelial contact guidance on well-defined micro- and nanostructured substrates. Journal of Cell Science 116(10), 1881 - 1892.
Thibault, M.M., Hoemann, C.D., and Buschmann, M.D., 2007. Fibronectin, vitronectin, and collagen I induce chemotaxis and haptotaxis of human and rabbit mesenchymal stem cells in a standardized transmembrane assay. Stem Cells and Development 16 (3), 489 - 502.
Titmarsh, D., Hidalgo, A., Turner, J., Wolvetang, E., and Cooper-White, J., 2011. Optimization of flowrate for expansion of human embryonic stem cells in perfusion microbioreactors. Biotechnology and Bioengineering 108(12), 2894 - 2904.
Tsai, T.H., Liou, D.S., Kuo, L.S., and Chen, P.H., 2009. Rapid mixing between ferro-nanofluid and water in a semi-active Y-type micromixer. Sensors and Actuators A 153(2), 267 - 273.
Wahl, S.M., Hunt, D.A., Wakefield, L.M., McCartney-Francis, N., Wahl, L.M., Roberts, A.B., and Sporn, M.B., 1987. Transforming growth factor type β induces monocyte chemotaxis and growth factor production. Proceedings of the National Academy of Sciences of the United States of America 84(16), 5788 - 5792.
Wallace, D.G., and Rosenblatt, J., 2003. Collagen gel systems for sustained delivery and tissue engineering. Advanced Drug Delivery Reviews 55(12), 1631 - 1649.
Wang, S. J., Saadi, W., Lin, F., Nguyen, C.M., and Jeon, N.L., 2004. Differential effects of EGF gradient profiles on MDA-MB-231 breast cancer cell chemotaxis. Experimental Cell Research 300(1), 180 - 189.
Webb, S.E., Pollard, J.W., and Jones, G.E., 1996. Direct observation and quantification of macrophage chemoattraction to the growth factor CSF-1. Journal of Cell Science 109, 793 - 803.
Weibel, D.B., Garstecki, P., and Whitesides, G.M., 2005. Combining microscience and neurobiology. Current Opinion in Neurobiology 15(5), 560 - 567.
Whitesides, G.M., and Stroock, A.D., 2001. Flexible methods for microfluidics. Physics Today 54(6), 42 - 48.
Willson Research Group, 2002. Post-Exposure Bake. http://willson.cm. utexas.edu/Research/Sub_Files/Resist_Modeling/peb.htm
Wu, H.Y., and Liu, C.H., 2005. A novel electrokinetic micromixer. Sensors and Actuators A 118(1), 107 - 115.
Xia, Y., and Whitesides, G.M., 1998. Soft Lithography. Angew. Chem. Int. Ed. 37, 550 - 575.
Yen, B.L., Huang, H.I., Chien, C.C., Jui, H.Y., Ko, B.S., Yao, M., Shun, C.T., Yen, M.L., Lee, M.C., and Chen, Y.C., 2005. Isolation of multipotent cells from human term placenta. Stem Cells 23(1), 3 - 9.
Zigmond, S.H., 1977. Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors. The Journal of Cell Biology 75 (2), 606 - 616.
陳誌遠，2012。利用博登量測器分析人類胎盤源多功能幹細胞的化學趨向行為。中央大學機械工程研究所，博士論文。
梅（May, Gary S.）著，林鴻志譯，2008。半導體製程概論。交大出版社。
黃蓉芬與楊燕枝，2003。微機電製程技術應用於生醫領域之研究。工業技術研究院產業經濟與資訊服務中心。
伍秀菁，汪若文，林美吟編輯，2009。微機電系統技術與應用。財團法人國家實驗研究院儀器科技研究中心。
行政院衛生署，2012。民國100年死因統計結果分析。行政院衛生署。

指導教授

鍾志昂(Chin-ang Chung)

審核日期

2013-8-5

推文