臺灣博碩士論文加值系統

本研究為開發灌流式三維細胞培養微流體生醫晶片應用於即時且非侵入式的細胞動態監測，此晶片可以在不同培養條件下評估細胞增殖或化療敏感度，在不犧牲培養細胞的情況下，直接檢測在三維細胞培養過程中的細胞數目或存活率。此生醫晶片之結構包括一基板，一覆蓋層和流體層，基板為玻璃材料且在其表面上製作一對三維垂直電極，覆蓋層和流體層是由聚二甲基矽氧烷（PDMS）材料和軟刻技術而製作形成，培養室是由覆蓋層的開口部分所定義，而流道層提供流道連接灌注培養液，最後三層互相結合，可以製造出微流體生醫晶片。至於三維垂直電極之製作，首先在基板上使用微製造技術製作出平面電極，然後，覆蓋層與基板結合，且對準培養室和平面電極。最後藉由電鍍銅過程，從平面電極生長出一對對立於培養室兩側的三維垂直電極。
在這研究中，使用人類口腔癌細胞株（OEC-M1）作為實驗用之細胞，細胞是封裝在三維瓊脂膠體支架內，並在微流體生醫晶片上進行培養。在三維培養結構中的細胞反應，包括細胞成長數目和存活率，可在晶片上直接進行阻抗測量來估計，因此，進行長達5天的灌流式三維細胞培養，且細胞增殖可以藉由阻抗分析來監測。此外，在不同濃度的抗癌藥物灌注下，進行即時細胞存活率監測，阻抗值與細胞的存活率有直接的相關性，從細胞存活率的終點分析可以確認，藥物濃度的效果可被顯示出來，並且利用此量測技術，在藥物治療下的細胞存活率反應可藉由阻抗測量作即時監測。這項研究表明，可以通過阻抗測量監視在三維細胞培養模式下的細胞增殖和化療敏感度，且晶片阻抗最小檢測極限為80個細胞，所以此微流體生醫晶片，具有很高的發展潛力去開發一個分析平台用於癌症研究。

A perfusion three dimensional (3D) cell culture microfluidic chip has been developed for the real-time and non-invasive impedimetric monitoring of cell dynamics. The chip can continuously estimate the cell proliferation or chemosensitivity under different culture conditions. It is capable of onsite detecting the cell number or viability in the 3D cell culture construct without sacrificing the cultured cells. The chip consists of a substrate, a cover layer and a fluidic layer. The chip substrate is a glass substrate and a pair of 3D vertical electrodes is fabricated on its surface. The cover layer and fluidic layer are made of poly¬dimethylsiloxane (PDMS) material and formed by soft lithography. A culture chamber is defined by the openings of the cover layer and the fluidic layer provides fluidic connection for medium perfusion purpose. By bonding these layers, the microfluidic chip can be fabricated. For the fabrication of the 3D vertical electrodes, planar electrodes were first fabricated on the substrate by standard micro-fabrication techniques. Then, the cover layer was bonded to the substrate with the alignment of the culture chamber and the planar electrodes. By copper electroplating process, a pair of 3D vertical electrodes were grown from the planar electrodes and located at the opposite sidewalls of the culture chamber.
In this study, human oral cancer cell-line (OEC-M1) were used and encapsulated in 3D agarose scaffold and cultured in the microfluidic chip. On-site impedance measurement was performed to estimate the cellular response, i.e., cell number and viability, in the 3D culture construct. Cell number in the 3D construct was shown to be proportional to the impedance magnitude of the entire construct. Therefore, perfusion 3D cell culture was performed for up to 5 days and cell proliferation can be monitored by the impedimetric analysis. Furthermore, real-time monitoring of cell viability under the perfusion of anti-cancer drug in different concentrations was conducted and the impedance magnitude was directly correlated with the cell viability. From the confirmation of the endpoint cell viability assays, a concentration-dependent effect was shown; however, the response of cell viability during the drug treatment was able to be traced by the impedance measurement. This work showed cell proliferation and chemosensitivity in 3D cell culture format can be monitored by impedance measurement. This microfluidic chip has a high potential to develop a useful analytical platform for cancer research.

目 錄
誌謝 …………………………………………………………IV
中文摘要 …………………………………………………………V
英文摘要 …………………………………………………………VII
目錄 …………………………………………………………IX
圖目錄 ……………………………………………………………XII
表目錄 ……………………………………………………………XV
第一章 緒論 …………………………………………………………1
1-1研究背景和動機…………………………………………………1
1-2 研究目的 ……………………………………………………2
第二章 文獻回顧 …………………………………………………3
2-1細胞培養相關文獻………………………………………………3
2-2電阻抗感測器相關文獻…………………………………………11
2-3 文獻總結….……………………………………………………17
第三章 生醫晶片設計及感測原理…………………………………19
3-1生醫晶片設計…………………………………………………19
3-2生醫晶片阻抗分析原理…………………………………………20
3-2-1細胞阻抗特性………………………………………………20
3-2-2細胞阻抗模型………………………………………………23
第四章 生醫晶片製程………………………………………………26
4-1製程設計……………………………………………………26
4-2製程介紹……………………………………………………29
4-2-1薄膜沉積…………………………………………………29
4-2-2黃光微影…………………………………………………30
4-2-3濕式蝕刻…………………………………………………31
4-2-4電鍍………………………………………………………32
4-2-5軟刻製程…………………………………………………33
第五章 細胞生長動態分析方法及流程…………………………35
5-1細胞載入和培養平台………………………………………35
5-2生物分析介紹…………………………………………………37
5-2-1細胞數目成長分析…………………………………………37
5-2-2細胞存活率分析(CCK-8 assay)……………………………38
5-2-3乳酸分析………………………………………………………39
5-2-4螢光分析………………………………………………………39
第六章 細胞生長動態結果與分析……………………………………40
6-1電阻抗量化三維細胞培養膠體中之細胞數目……………40
6-2即時動態三維細胞培養膠阻抗量化 ……………………………44
6-3即時藥物濃度之細胞存活率之阻抗觀察…………………50
第七章 結論…………………………………………………………55
參考文獻…………………………………………………………………56
發表著作………………………………………………………………64
附錄 投稿文章…………………………………………………………66
圖 目 錄
圖2-1 細胞培養液灌流培養示意圖……………………………………4
圖 2-2 二維細胞培養和三維細胞培養的比較圖………………………5
圖2-3 腫瘤細胞之間的相互作用………………………………………7
圖2-4 無凝膠的微流體培養系統………………………………………8
圖2-5 二維微流體細胞培養晶片………………………………………9
圖2-6 三維微流體細胞培養晶片……………………………………..10
圖2-7 軟刻製程…………………………………………………………11
圖2-8 生醫晶片基底材料………………………………………………13
圖2-9 矽基板生物晶片…………………………………………………13
圖2-10 微流體納米指插電極陣列………………………………………14
圖2-11 二維細胞培養阻抗變化…………………………………………15
圖2-12 嵌入式黃金指叉電極 ………………………………………16
圖2-13 大腸桿菌O157：H7檢測晶片………………………………16
圖2-14 二維細胞阻抗原理圖………………………………………………17
圖3-1 生醫晶片分解圖………………………………………………20
圖3-2 細胞膜構造………………………………………………………21
圖3-3 通過細胞的電流圖…………………………………………22
圖3-4 二維電極電場和三維電極電場示意圖………………………24
圖3-5 三維環境之生醫晶片等效電路圖……………………………24
圖4-1 生醫晶片二維與三維電極製作圖………………………………28
圖4-2 流道製程圖……………………………………………………29
圖4-3 黃光微影製程步驟………………………………………………31
圖4-4 濕式蝕刻製程步驟……………………………………………32
圖4-5 電鍍架設圖…………………………………………………………33
圖4-6 三維電極完成圖………………………………………………33
圖4-7 軟刻技術製程步驟…………………………………………………34
圖4-8 PMMA模具………………………………………………………34
圖5-1 實驗過程中灌注三維細胞培養…………………………………36
圖5-2 灌流式細胞培養架構…………………………………………36
圖5-3 灌流式細胞培養晶片圖………………………………………36
圖5-4 WST8和WST8 formazan之結構…………………………………38
圖6-1 生醫晶片電阻抗分析…………………………………………40
圖6-2 不同頻率和細胞濃度阻抗之關係……………………………41
圖6-3 控制組和細胞濃度106 cell/ml阻抗比較圖…………………42
圖6-4 生醫晶片相位角分析…………………………………………43
圖6-5 不同細胞數量和細胞阻抗之關係………………………………44
圖6-6 5天DNA細胞成長百分比………………………………………46
圖6-7 生醫晶片細胞培養5天電阻抗分析………………………………47
圖6-8 電阻抗分析和細胞數目成長分析之關係…………………………48
圖6-9 生醫晶片乳酸分析…………………………………………………49
圖6-10螢光顯微鏡下觀察細胞活性…………………………………49
圖6-11即時抗癌藥物濃度細胞阻抗圖……………………………………51
圖6-12抗癌藥物濃度和細胞存活率之關係.……………………………52
圖6-13不同藥物濃度共軛焦顯微鏡螢光死活圖…………………………53
圖 6-14細胞阻抗和細胞存活率之關係…………………………………54
表目錄
表2-1 細胞培養比較表…………………………………………………18

參考文獻
[1] D.C. Duffy, J.C. McDonald, O.J.A. Schueller and G.M. Whitesides, "Rapid Prototyping of Microfluidic Systems in Poly (dime¬thylsilo¬xance)", Analytical Chemistry, 70, pp. 4974-4984, 1998.
[2] M.A. Unger, H.P. Chou, T. Thorsen, A. Scherer and S.R. Quake, "Monolithic Micr¬ofabricated Valves and Pumps by Multilayer Soft Lithography", Science, 288, pp. 113-116, 2000.
[3] H. Andersson and A.V.D. Berg, "Microfluidic devices for cell-omics : a review", Sensors and Actuators B: Chemical, 92, pp. 315-325, 2003.
[4] J.W. Haycock, "3D Cell Culture: A Review of Current Aproaches and Techniques", Methods in Molecular Biology, 695, pp. 1-15, 2010.
[5] A. Abbot, "Cell culture: biology's new dimension", Nature, 424, pp. 870-872, 2003.
[6] J. Lee, M.J. Cuddihy and N.A. Kotov, "Three-Dimensional Cell Culture Matrices: State of the Art", Tissue Engineering Part B: Review, 14, pp.61-86, 2008.
[7] M.H. Wu, S.B. Huang, Z. Cui, Z. Cui and G.B. Lee, "Development of perfusion-based micro 3-D cell culture platform and its application for high throughput drug testing", Sensors and Actuators B: Chemical, 129, pp. 231-240, 2008.
[8] M. Sittinger, O. Schultz, G. Keyszer, W.W. Minuth and G.R. Burmester, "Artificial tissues in perfusion culture", The Interna-tional Journal of Artificial Organs, 20, pp. 57-62,1997.
[9] K. Ziolkowska, E. Jedrych, R. Kwapiszewski, J. Lopacinska, M. Skolimowski and M. Chu, "PDMS/ glass microfluidic cell culture system for cytotoxicity tests and cells passage", Sensors and Actuators B: Chemical, 145, pp. 533-542, 2010.
[10] M.H. Wu, S.B. Huang and G.B. Lee, "Microfluidic cell culture systems for drug research", Lab on a Chip, 10, pp. 939–956, 2010.
[11] K. Ziolkowska, E. Jedrych, R. Kwapiszewski, J. Lopacinska, M. Skolimowski and M. Chudy, "PDMS/glass microfluidic cell cul-ture system for cytotoxicity tests and cells passage", Sensors and Actuators B: Chemical, 145, pp. 533-542, 2010.
[12] L. Kim, Y.C. Toh, J. Voldman and H. Yu, "A practical guide to microfluidic perfusion culture of adherent mammalian cells", Lab on a Chip, 7, pp. 164-167, 2007.
[13] E.A. Jamil, P.K. Sorger and K.F. Jensen, "Cells on chips", Nature, 442, pp. 403-411, 2006.
[14] L.G. Griffith and M.A. Swartz, "Capturing complex 3D tissue physiology in vitro", Nature reviews. Molecular cell biology, 7, pp. 211-224, 2006.
[15] E. Leclerc, Y. Sakai and T. Fujii, "Cell Culture in 3-Dimensional Microfluidic Structure of PDMS (polydimethylsiloxane)", Biomedical Microdevices, 5, pp. 109-114, 2003.
[16] C. Zhang, S.M. Chia, S.M. Ong, S. Zhang, Y.C. Toh, D.V. Noort and H. Yu, "The controlled presentation of TGF-b1 to hepatocytes in a 3D-microfluidic cell culture system", Biomaterials, 30, pp. 3847-3853, 2009.
[17] S.M. Ong, C. Zhang, Y.C. Toh, S.H. Kim, H.L. Foo, C.H. Tan, D.V. Noort, S. Park and H. Yu, "A gel-free 3D microfluidic cell culture system", Biomaterials, 29, pp. 3237-3244, 2008.
[18] M.K. Hulvey and R.S. Martin, "A microchip-based endothelium mimic utilizing open reservoirs for cell immobilization and integrated carbon ink microelectrodes for detection", Analytical and Bioanalytical Chemistry, 393, pp. 599-605, 2009.
[19] B.J. Kane, M.J. Zinner, M.L. Yarmush and M. Toner, "Liv-er-Specific Functional Studies in a Microfluidic Array of Primary Mammalian Hepatocytes", Analytical Chemistry, 78, pp. 4291-298, 2006.
[20] A. Sivaraman, J.K. Leach, S. Townsend, T. Iida, B.J. Hogan, D.B. Stolz, R. Fry, L.D. Samson, S.R. Tannenbaum and L.G. Griffith, "A Microscale In Vitro Physiological Model of the Liver: Predictive Screens for Drug Metabolism and Enzyme Induction", Current Drug Metabolism, 6, pp. 569-591, 2005.
[21] S.K. Sia and G.M. Whitesides, "Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies", Electrophoresis, 24, pp. 3563-3576, 2003.
[22] P.S. Dittrich and A. Manz, "Lab-on-a-chip: microfluidics in drug discovery", Nature Reviews Drug Discovery, 5, pp. 210-218, 2006.
[23] P.J. Hung, J. Lee, P. Sabounchi, N. Aghdam, R. Lin and L.P. Lee, "A novel high aspect ratio microfluidic design to provide a stable and uniform microenvironment for cell growth in a high throughput mammalian cell culture array", Lap on a chip, 5, pp. 44-48, 2005.
[24] J.T. Nevill, R. Cooper, M. Dueck, D.N. Breslauer and L.P. Lee, "Integrated microfluidic cell culture and lysis on a chip", Lap on a chip, 7, pp. 1689-1695, 2007.
[25] A. Shamloo, N. Ma, M.M. Poo, L.L. Sohn and S.C. Heilshorn, "Endothelial cell polarization and chemotaxis in a microfluidic device", Lap on a chip, 8, pp. 1292-1299, 2007.
[26] Y.S. Torisawaa, H. Shikua, T. Yasukawaa, M. Nishizawab and T. Matsuea, "Three-dimensional micro-culture system with a silicon-based cell array device for multi-channel drug sensitivity test", Sensors and Actuators B: Chemical, 108, pp. 654-659, 2005.
[27] J. Lii, W.J. Hsu, H. Parsa, A. Das, R. Rouse and S.K. Sia, "Re-al-Time Microfluidic System for Studying Mammalian Cells in 3D Microenvironments", Analytical Chemistry, 80, pp. 3640-3647, 2006.
[28] S.B. Huang, M.H. Wu, S.S. Wang and G.B. Lee, "Microfluidic cell culture chip with multiplexed medium delivery and efficient cell/scaffold loading mechanisms for high-throughput perfusion 3-dimensional cell culture-based assays", Biomedical Microdevices, 13, pp. 415-430, 2011.
[29] N. Bao, J. Wang and C. Lu, "Recent advances in electric analysis of cells in microfluidic systems", Analytical and Bioanalytical Chemistry, 391, pp. 933-942, 2008.
[30] Y. Cho, H.S. Kim and A.B. Frazier, "Whole-Cell Impedance Analysis for Highly and Poorly Metastatic Cancer Cells", Journal of Microelectromechanical Systems, 18, pp. 808-817, 2009.
[31] J.H. Yeon and J.K. Park, "Cytotoxicity test based on electrochemical impedance measurement of HepG2 cultured in micr¬ofabricated cell chip", Analytical Biochemistry, 341, pp. 308-315, 2005.
[32] G. Kang, S.K. Lee, S.K. Yoo, S. Yang and J.H. Lee, "Impedance Measurement of Normal and Cancerous Human Breast Cells Using a Microfluidic Tunnel", Sensors IEEE, pp. 2109- 2112, 2010.
[33] P. Silley, "Impedance microbiology-a rapid change for microbiologists", Journal of Applied Bacteriology, 80, pp. 233-243, 1996.
[34] A.G.E. Saum, R.H. Cumming and F.J. Rowell, "Use of substrate coated electrodes and AC impedance spectroscopy for the detection of enzyme activity", Biosensors and Bioelectronics, 13, pp. 511-518, 1998.
[35] S. Grant, F. Davis, K.A. Law, A.C. Barton, S.D. Collyer, S.P.J. Higson and T.D. Gibson, "Label-free and reversible immunosensor based upon an ac impedance interrogation protocol", Analytica Chimica Acta, 537 , pp. 163-168, 2005.
[36] K.S. Ma, H. Zhou, J. Zoval and M. Madou, "DNA hybrid-ization detection by label free versus impedance amplifying label with impedance spectroscopy", Sensors and Actuators B: Chemical, 114 , pp. 58-64, 2006.
[37] N.N. Mishra, S. Retterer, T.J. Zieziulewicz, M. Isaacson, D. Szarowski, D.E. Mousseau, D.A. Lawrence and J.N. Turner, "On-chip micro-biosensor for the detection of human CD4+ cells based on AC impedance and optical analysis", Biosensors and Bioelectronics, 21, pp. 696-704, 2005.
[38] N.M. Nelson, D. Sander and P. Abshire, "A Cell Impedance Sensor based on a Silicon Cochlea", Biomedical Circuits and Systems Conference, pp. 117 -120, 2009.
[39] Z. Zou, J. Kai, M.J. Rust, J. Han and C.H. Ahn, "Functionalized nano interdigitated electrodes arrays on polymer with integrated microfluidics for direct bio-affinity sensing using impedimetric measurement", Sensors and Actuators A: Physical, 136, pp. 518-526, 2007.
[40] P. Linderholm, T. Braschler, J. Vannod, Y. Barrandon, M. Brouard and P. Renauda, "Two　dimen¬sion¬al impedance imaging of cell migration and epithelial stratification" , Lab on a chip, 6, pp. 1155-1162, 2006.
[41] H. Slanina, A. König, H. Claus, M. Frosch and A.S. Unkmeir, "Real-time impedance analysis of host cell response to meningococcal infection", Journal of Microbiological Methods, 84, pp. 101-108, 2011.
[42] M. Varshney, Y. Li, B. Srinivasan and S. Tung, "A label-free microfluidics and interdigitated array microelectrode-based impedance biosensor in combination with nanoparticles immuno¬separation for detection of Escherichia coli O157:H7 in food samples", Sensors and Actuators B: Chemical, 128, pp. 99-107, 2007.
[43] S. Diemert, A.M. Dolga, S. Tobaben, J. Grohm, S. Pfeifer, E. Oexler and C. Culmsee, "Impedance measurement for real time detection of neuronal cell death", Journal of Neuroscience Meth-ods, 203, pp. 69-77, 2012.
[44] 游順生，〈電阻抗分析應用於細胞凋亡量化評估〉，國立成功大學醫學工程研究所，碩士論文，民國98年。
[45] R. Pethig, "Dielectric properties of biological materials : bio-physical and medical applications", IEEE Transactions on Elec-trical Insulation, EI-19, pp. 453-474, 1984.
[46] 李孝貞〈以阻抗分析系統評估維生素 C 處理下所影響之細胞修復行為〉，國立成功大學醫學工程研究所，碩士論文，民國98年。
[47] I. Giaever and C. Keese, "Micromotion of mammalian cells measured electrically", Proceedings of the National Academy of Sciences, 88, pp. 7896-7900, 1991.
[48] B.W. Chang, C.H. Chen, S.J. Ding, D.C.H. Chen and H.C. Chang, "Impedimetric monitoring of cell attachment on interdigitated microelectrodes", Sensors and Actuators B: Chemical, 105, pp. 159-163, 2005.
[49] Y. Qiu, R. Lia and X. Zhang, "Real-Time Monitoring Primary Cardiomyocyte Adhesion Based on Electrochemical Impedance Spectroscopy and Electrical Cell-Substrate Impedance Sensing", Analytical Chemistry, 80, pp. 990-996, 2008.
[50] C.M. Lo, C.R. Keese and I. Giaever, "Impedance Analysis of MDCK Cells Measured by Electric Cell-Substrate Impedance Sensing", Biophysical Journal, 69, pp. 2800-2807.
[51] C.M. Lo and J. Ferrier, "Impedance analysis of broblastic cell layers measured by electric cell-substrate impedance sensing", Physical Review E, 57, pp. 6982-6987, 1988.
[52] S. Grimnes and O.G. Martinsen, Bioimpedance and bioelectricity basics. Academic Press, 2000.
[53] H.G.L. Coster, T.C. Chilcott and A.C.F. Coster, "Impedance spectros¬copy of interfaces, membranes and ultrastructures", Bioelectro¬chemistry and Bioenergetics, 40, pp. 79-98, 1998.
[54] A.J. Bard and L.R. Faulkner, Electrochemical methods: funda-mentals and applications, Wiley, New York, 2001.
[55] 高敏峯〈單一細胞阻抗平台之研發〉，國立成功大學電機工程 研究所，碩士論文，民國96年。