藉由微流道系統量測細胞於帶電表面之貼附力

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姓名

魏瑋昕(Wei-shin Wei) 查詢紙本館藏

畢業系所

生物醫學工程研究所

論文名稱

藉由微流道系統量測細胞於帶電表面之貼附力
(Measurement of Cell Adhesive Force on Charged Surface by Multiple-Channel Microfluidic Device)

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摘要(中)

本研究主要以剪應力計算細胞與不同材料表面間的貼附能力，進一步提出細胞純化的指標方法。首先，我們合成一系列雙性高分子，其親水部分由完全攜帶負電荷，負電荷與雙離子(zwitterionic)混合，或全然為雙離子的結構所組成。我們將二甲氨基丙胺(3-(Dimethylamino)-1-propylamine, DMAPA)與乙胺(Ethylamine, EA)以不同比例混合，然後與聚2,5-呋喃二酮-1-十八烯(Poly(maleic anhydride-alt-1-octadecene), MAO)上的maleic anhydride進行開環，可形成一具有長碳鏈疏水片段，帶羧酸基及雙離子性結構的共聚高分子。此高分子一方面可藉由其疏水片段吸附於聚二甲基矽氧烷(Polydimethylsiloxane, PDMS)基材，另一方面亦可藉由曝露在外的負電荷與雙離子性官能基來改變細胞貼附力。我們選用的細胞為大鼠骨髓間葉幹細胞(Sprague-Dawley rat bone marrow stem cells, RM )、小鼠胚胎纖維母細胞 (Mouse embryonic fibroblast cell line, NIH-3T3)與人類纖維腫瘤(HT1080)。細胞經12小時培養後，發現三種細胞於PDMS表面上的細胞型態多為梭長及多角型，而在親水部分全為雙離子結構的MAO-DMAPA-EA1:8:0表面，則多為圓型。當反應時EA比例逐漸增加，也就是負電荷逐漸增加時，細胞貼附型態由圓型轉為梭長或多角型，且細胞貼附量也隨EA比例增加而增多。進一步，我們將所合成之共聚高分子塗佈於微流道內，改變流速，進行細胞貼附力的量化測試，可以得知不同細胞在不同表面的附著力，從而設計出細胞純化篩選的方法。

摘要(英)

In this study, we coated the amphipathic charged polymers on polydimethylsiloxane (PDMS) surface for cell adhesion. To examine the cell adhesion force, the multiple-channel microfluidic device was used. The ring opening reaction between poly(maleic anhydride-alt- 1-octadecene (MAO) and different molar ratio of ethylamine (EA) and 3-(Dimethylamino)-1-propylamine (DMAPA) were carried out. From the analysis of NMR spectrum, we obtain the MAO:DMAPA=1:8 with all maleic anhydride functional groups being 100 % ring opening and this polymer possesses zwitterionic functional group in co-polymer at pH 7.4. For MAO-DMAPA-EA co-polymer synthesis, we found the content of zwitterionic functional groups would decrease with increasing the addition of EA, and the negatively charged groups would also increase in co-polymer. After coating the co-polymers with different charged group content on PDMS surface, the hydrophilicity of surface and anti-protein adsorption ability would increase as the zwitterionic functional groups increase on co-polymer. Furthermore, the primary cell sprague-dawley rat bone marrow stem cells (RM), mouse embryonic fibroblast cell line (NIH-3T3), and human sarcoma cell line (HT1080) were cultured on co-polymer coated surface for 12 hr. We found that the morphology of adhesive NIH-3T3 cells was spindle-like and polygonal on PDMS surface and spherical shape on MAO-DMAPA-EA 1:8:0 coated surface. With the increase of EA content on co-polymer, the morphology of cellular adhesion turned from spherical into spindle-like and polygonal. The amount of cellular adhesion increased with the ethylamine composition increase. Similar results were also observed on RM and NIH3T3 cells adhesion. We obtain the shear forces of RM on the co-polymer surface are ranging from 0 to 4.62 dyne/cm2. The shear stress of cell adhesion is related to the zwittionic content on co-polymer, that is, the high zwitterionic content would result in low adhesion force.

關鍵字(中)

★ 聚二甲基矽氧烷
★ 微流道
★ 雙離子性
★ 大鼠骨髓間葉幹細胞
★ 剪應力

關鍵字(英)

★ Polydimethylsiloxane, PDMS
★ microfluidic
★ zwitterionic
★ Sprague-Dawley rat bone marrow stem cells
★ shear stress

論文目次

目錄
摘要 I
Abstract III
圖目錄 VIII
表目錄 XI
第一章緒論 1
1.1研究動機 1
1.2研究目的 2
第二章文獻回顧 4
2.1 細胞類型及貼附機制 4
2.1.1細胞類型 4
2.1.2 細胞貼附機制 4
2.2 細胞分選 8
2.2.1常見的細胞分選方式 10
2.3微流體晶片之簡介 17
2.3.1微流道之材料 18
2.3.4 PDMS Microchip 的表面修飾方法 19
2.4生物分子與材料表面之交互作用 23
2.4.1 蛋白質與材料表面作用之機制 23
2.4.2雙離子性高分子 25
第三章實驗藥品、儀器設備與方法 30
3.1實驗藥品及儀器設備30
3.1.1兩性雙離子性高分子製備及PDMS表面改質 30
3.1.2 微流體晶片製程及實驗儀器 32
3.1.3 細胞培養 33
3.2 實驗方法 34
3.2.1實驗架構 34
3.2.2 兩性雙離子性高分子之製備及性質鑑定 35
3.2.3 PDMS表面改質方法及表面性質測定 37
3.2.4 微流體晶片製程 39
3.2.5細胞培養 47
第四章結果與討論 52
4.1兩性雙離子性共聚高分子之合成 52
4.1.1 MAO-DMAPA-EA高分子之合成 52
4.2 兩性雙離子性高分子之結構與物化特性 54
4.2.1 MAO-DMAPA-EA高分子之結構鑑定 54
4.3 MAO-DMAPA-EA之物化性質 58
4.3.1 MAO-DMAPA-EA之pKa之量測 58
4.3.2 MAO-DMAPA-EA之溶解性 60
4.4 MAO-DMAPA-EA在PDMS表面之塗佈 61
4.4.1量測MAO-DMAPA-EA塗佈表面之親疏水性 61
4.4.2高分子塗佈於PDMS基材表面之元素分析 67
4.4.3蛋白質於高分子塗佈表面的吸附行為 68
4.5 細胞於MAO-DMAPA-EA塗佈表面之貼附行為 70
4.6 流場剪應力對細胞貼附力之影響 75
4.6.1晶片內各培養區的細胞均勻度 75
4.6.2微流道與流場剪應力之關係 77
第五章結論 85
參考文獻 87

參考文獻

1. Preston, S. L.; Alison, M. R.; Forbes, S. J.; Direkze, N. C.; Poulsom, R.; Wright, N. A., The new stem cell biology: something for everyone. Molecular pathology : MP 2003, 56 (2), 86-96.
2. Ghafar-Zadeh, E.; Waldeisen, J. R.; Lee, L. P., Engineered approaches to the stem cell microenvironment for cardiac tissue regeneration. Lab on a chip 2011, 11 (18), 3031-48.
3. Didar, T. F.; Tabrizian, M., Adhesion based detection, sorting and enrichment of cells in microfluidic Lab-on-Chip devices. Lab on a chip 2010, 10 (22), 3043-53.
4. Becker, H.; Locascio, L. E., Polymer microfluidic devices. Talanta 2002, 56 (2), 267-287.
5. Pierschbacher, M. D.; Ruoslahti, E., Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 1984, 309 (5963), 30-3.
6. Anselme, K., Osteoblast adhesion on biomaterials. Biomaterials 2000, 21 (7), 667-81.
7. Nilsson, K., Microcarrier cell culture. Biotechnology & genetic engineering reviews 1988, 6, 403-39.
8. Sigrist, H.; Collioud, A.; Clemence, J. F.; Gao, H.; Luginbuhl, R.; Sanger, M.; Sundarababu, G., Surface Immobilization of Biomolecules by Light. Opt Eng 1995, 34 (8), 2339-2348.
9. Neild, A.; Ng, T. W.; Yii, W. M., Optical sorting of dielectric Rayleigh spherical particles with scattering and standing waves. Optics express 2009, 17 (7), 5321-9.
10. Yang, A. H.; Soh, H. T., Acoustophoretic sorting of viable mammalian cells in a microfluidic device. Analytical chemistry 2012, 84 (24), 10756-62.
11. Augustsson, P.; Magnusson, C.; Nordin, M.; Lilja, H.; Laurell, T., Microfluidic, label-free enrichment of prostate cancer cells in blood based on acoustophoresis. Analytical chemistry 2012, 84 (18), 7954-62.
12. Khoshmanesh, K.; Nahavandi, S.; Baratchi, S.; Mitchell, A.; Kalantar-zadeh, K., Dielectrophoretic platforms for bio-microfluidic systems. Biosensors & bioelectronics 2011, 26 (5), 1800-14.
13. Yang, F.; Yang, X.; Jiang, H.; Bulkhaults, P.; Wood, P.; Hrushesky, W.; Wang, G., Dielectrophoretic separation of colorectal cancer cells. Biomicrofluidics 2010, 4 (1), 13204.
14. Doh, I.; Cho, Y.-H., A continuous cell separation chip using hydrodynamic dielectrophoresis (DEP) process. Sensors and Actuators A: Physical 2005, 121 (1), 59-65.
15. Jen, C.-P.; Huang, C.-T.; Weng, C.-H., Focusing of biological cells utilizing negative dielectrophoretic force generated by insulating structures. Microelectronic Engineering 2010, 87 (5-8), 773-777.
16. Li, J. M.; Liu, C.; Dai, X. D.; Chen, H. H.; Liang, Y.; Sun, H. L.; Tian, H.; Ding, X. P., PMMA microfluidic devices with three-dimensional features for blood cell filtration. Journal of Micromechanics and Microengineering 2008, 18 (9), 095021.
17. Chen, X.; Cui, D.; Liu, C.; Li, H.; Chen, J., Continuous flow microfluidic device for cell separation, cell lysis and DNA purification. Anal Chim Acta 2007, 584 (2), 237-43.
18. Inokuchi, H.; Suzuki, Y.; Kasagi, N.; Shikazono, N.; Furukawa, K.; Ushida, T., Micro Magnetic Separator for Stem Cell Sorting System. 2005.
19. Ito, A.; Shinkai, M.; Honda, H.; Kobayashi, T., Medical application of functionalized magnetic nanoparticles. Journal of bioscience and bioengineering 2005, 100 (1), 1-11.
20. Pamme, N., Continuous flow separations in microfluidic devices. Lab on a chip 2007, 7 (12), 1644-59.
21. Adams, J. D.; Kim, U.; Soh, H. T., Multitarget magnetic activated cell sorter. Proceedings of the National Academy of Sciences of the United States of America 2008, 105 (47), 18165-70.
22. Studer, V., A microfluidic mammalian cell sorter based on fluorescence detection. Microelectronic Engineering 2004, 73-74, 852-857.
23. Wang, M. M.; Tu, E.; Raymond, D. E.; Yang, J. M.; Zhang, H.; Hagen, N.; Dees, B.; Mercer, E. M.; Forster, A. H.; Kariv, I.; Marchand, P. J.; Butler, W. F., Microfluidic sorting of mammalian cells by optical force switching. Nature biotechnology 2005, 23 (1), 83-7.
24. Ng, C. P.; Pun, S. H., A perfusable 3D cell-matrix tissue culture chamber for in situ evaluation of nanoparticle vehicle penetration and transport. Biotechnology and bioengineering 2008, 99 (6), 1490-501.
25. Ong, S. M.; Zhang, C.; Toh, Y. C.; Kim, S. H.; Foo, H. L.; Tan, C. H.; van Noort, D.; Park, S.; Yu, H., A gel-free 3D microfluidic cell culture system. Biomaterials 2008, 29 (22), 3237-44.
26. Manz, A.; Graber, N.; Widmer, H. M., MINIATURIZED TOTAL CHEMICAL-ANALYSIS SYSTEMS - A NOVEL CONCEPT FOR CHEMICAL SENSING. Sensors and Actuators B-Chemical 1990, 1 (1-6), 244-248.
27. Miura, M.; Gronthos, S.; Zhao, M.; Lu, B.; Fisher, L. W.; Robey, P. G.; Shi, S., SHED: stem cells from human exfoliated deciduous teeth. Proceedings of the National Academy of Sciences of the United States of America 2003, 100 (10), 5807-12.
28. Pittenger, M. F., Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science 1999, 284 (5411), 143-147.
29. Chiem, N. H.; Harrison, D. J., Microchip systems for immunoassay: an integrated immunoreactor with electrophoretic separation for serum theophylline determination. Clin Chem 1998, 44 (3), 591-8.
30. Goddard, J. M.; Hotchkiss, J. H., Polymer surface modification for the attachment of bioactive compounds. Progress in Polymer Science 2007, 32 (7), 698-725.
31. Bhattacharya, S.; Datta, A.; Berg, J. M.; Gangopadhyay, S., Studies on surface wettability of poly(dimethyl) siloxane (PDMS) and glass under oxygen-plasma treatment and correlation with bond strength. Journal of Microelectromechanical Systems 2005, 14 (3), 590-597.
32. Chumbimuni-Torres, K. Y.; Coronado, R. E.; Mfuh, A. M.; Castro-Guerrero, C.; Silva, M. F.; Negrete, G. R.; Bizios, R.; Garcia, C. D., Adsorption of proteins to thin-films of PDMS and its effect on the adhesion of human endothelial cells. Rsc Adv 2011, 1 (4), 706-714.
33. McDonald, J. C.; Whitesides, G. M., Poly(dimethylsiloxane) as a material for fabricating microfluidic devices. Accounts Chem Res 2002, 35 (7), 491-499.
34. PITT, W. G., Fabrication of a Continuous Wettability Gradient by Radio Frequency Plasma Discharge. Journal of Colloid andlnterface Science 1989, Vol. 133, No. 1, , 223-227.
35. Wang, L.; Sun, B.; Ziemer, K. S.; Barabino, G. A.; Carrier, R. L., Chemical and physical modifications to poly(dimethylsiloxane) surfaces affect adhesion of Caco-2 cells. Journal of Biomedical Materials Research Part A 2010, 93A (4), 1260-1271.
36. Ting, L. H.; Feghhi, S.; Han, S. J.; Rodriguez, M. L.; Sniadecki, N. J., Effect of Silanization Film Thickness in Soft Lithography of Nanoscale Features. Journal of Nanotechnology in Engineering and Medicine 2011, 2 (4), 041006.
37. Wong, I.; Ho, C. M., Surface molecular property modifications for poly(dimethylsiloxane) (PDMS) based microfluidic devices. Microfluidics and nanofluidics 2009, 7 (3), 291-306.
38. Chen, Z. P.; Ren, W. C.; Gao, L. B.; Liu, B. L.; Pei, S. F.; Cheng, H. M., Three-dimensional flexible and conductive interconnected graphene networks grown by chemical vapour deposition. Nat Mater 2011, 10 (6), 424-428.
39. Brown, X. Q.; Ookawa, K.; Wong, J. Y., Evaluation of polydimethylsiloxane scaffolds with physiologically-relevant elastic moduli: interplay of substrate mechanics and surface chemistry effects on vascular smooth muscle cell response. Biomaterials 2005, 26 (16), 3123-3129.
40. Dechene, J. M., Surface Modifications of Poly(dimethylsiloxane) for Biological Applications of Microfluidic Devices. 2010.
41. Roman, G. T.; Culbertson, C. T., Surface engineering of poly(dimethylsiloxane) microfluidic devices using transition metal sol-gel chemistry. Langmuir 2006, 22 (9), 4445-4451.
42. Liu, Y.; Fanguy, J. C.; Bledsoe, J. M.; Henry, C. S., Dynamic coating using polyelectrolyte multilayers for chemical control of electroosmotic flow in capillary electrophoresis microchips. Analytical chemistry 2000, 72 (24), 5939-5944.
43. LenhoP, C. M. R. a. A. M., Electrostatic and van der Waals Contributions to Protein Adsorption: Comparison of Theory and Experiment. 1995.
44. Arakawa, T.; Tokunaga, M., Electrostatic and hydrophobic interactions play a major role in the stability and refolding of halophilic proteins. Protein Peptide Lett 2004, 11 (2), 125-132.
45. Tan, J. S.; Martic, P. A., Protein Adsorption and Conformational Change on Small Polymer Particles. J Colloid Interf Sci 1990, 136 (2), 415-431.
46. George S. Georgiev, E. B. K., Elena D. Vassileva, Irena P. Kamenova,; Ventsislava T. Georgieva, S. B. I., and Ivo A. Ivan, Self-Assembly, Antipolyelectrolyte Effect, and Nonbiofouling Properties of Polyzwitterions. 2006.
47. Lewis, A. L., Phosphorylcholine-based polymers and their use in the prevention of biofouling. Colloid Surface B 2000, 18 (3-4), 261-275.
48. Chang, Y.; Chen, S. F.; Zhang, Z.; Jiang, S. Y., Highly protein-resistant coatings from well-defined diblock copolymers containing sulfobetaines. Langmuir 2006, 22 (5), 2222-2226.
49. Zhang, Z.; Chen, S. F.; Chang, Y.; Jiang, S. Y., Surface grafted sulfobetaine polymers via atom transfer radical polymerization as superlow fouling coatings. J Phys Chem B 2006, 110 (22), 10799-10804.
50. Zhang, Z.; Vaisocherova, H.; Cheng, G.; Yang, W.; Xue, H.; Jiang, S., Nonfouling behavior of polycarboxybetaine-grafted surfaces: structural and environmental effects. Biomacromolecules 2008, 9 (10), 2686-92.
51. Zhang, Z.; Chao, T.; Chen, S.; Jiang, S., Superlow fouling sulfobetaine and carboxybetaine polymers on glass slides. Langmuir 2006, 22 (24), 10072-7.
52. Nagy, J. K.; Hoffmann, A. K.; Keyes, M. H.; Gray, D. N.; Oxenoid, K.; Sanders, C. R., Use of amphipathic polymers to deliver a membrane protein to lipid bilayers. Febs Lett 2001, 501 (2-3), 115-120.
53. 區理函, Hydrophobic surfaces coated with amphiphilic and zwitterionic polymers for biofouling resistance. 2011.
54. 陳威任, Study on the Mechanical Behavior of Madin-Darby Canine Kiney (MDCK) Epithelial Cell by Micro-Pillars Array. 2007.
55. Long-Sun Huang, P.-C., Tsai, The effects of microfluidic shear stress on endothelial differentiation of amniotic fluid stem cells. 2010.

指導教授

阮若屈(Ruoh-Chyu, Ruaan)

審核日期

2013-8-28

推文