細胞融合等技術目前被廣泛應用於基因重組、製造單株抗體、癌症免疫療法以及組織再生等各項生物應用，而現今細胞融合可利用生物、化學與物理方法達成。但是在傳統大型系統上，由於細胞間的不穩定接觸與隨機配對上的限制使得效率與產量上難以提升。因此，有效的增加細胞接觸與配對是相當關鍵之因素來提升細胞融合的效率與產量。本研究提出一新方法，透過整合微結構與光誘導技術於微流體系統來穩定細胞間的接觸與配對以達到精確、高效率、高產量的細胞融合。而光誘導技術是將設計好之圖形利用投影機將其投射在一鍍有非晶矽薄膜的氧化銦錫玻璃上，被照射之非金矽薄膜區域的導電度會提升數個量級，並同時在上下兩側之氧化銦錫玻璃上導入交流電場，如此一來在受光照射之非晶矽薄膜上之會產生局部之加強電場，進而形成”虛擬電極”，藉此來取代傳統系統上的固定微電極，使得能輕易且彈性的操縱電極之數量、大小、位置等參數來提升效率與產量，此虛擬電極已經成功地應用於細胞分離、細胞裂解、細胞電穿孔等應用。故本研究創造一新方法，先利用微流道上之微結構所產生之流場將細胞依序抓取且配對，並達到57%的配對率，再透過整合光誘導技術之虛擬電極使細胞融合效率達到87%。因此，光誘導細胞融合系統是一項可以進行高效率與高產量之細胞融合的先進技術。

Cell fusion is a critical course for all sort of biomedical applications including cell reprogramming, hybridoma formation, cancer immunotherapy, and tissue regeneration. It can be realized by using biological, chemical, or physical methods. However, efficiency and yields are limited by unstable cell contact and random cell pairings in traditional methods. Hence, improving cell contact and cell pairing are the two key factors to enhance efficiency and yields of cell fusion. This study therefore reported a new approach called optically-induced cell fusion (OICF) which integrates cell-pairing microstructures and optically-induced, localized electrical field to achieve precise cell fusion with high yields and high efficiency. By projecting light patterns on a photoconductive film (hydrogen-rich amorphous silicon, a-Si: H) coated on an indium-tin-oxide (ITO) glass while an alternating-current (AC) electric field was applied on the top and bottom ITO glasses, “virtual” electrodes would be constructed accordingly. In fact, this method could be used on several biomedical applications, including cell manipulation, cell separation, cell lysis and electroporation. Therefore, a locally enhanced electric field would be induced and the pairing cells could be precisely fused by the virtual electrodes. In this study, 57% cell paring rate and 87% fusion efficiency were achieved. Therefore, OICF is a promising method to succeed in cell fusion with high efficiency and high yields.

Abstract II
摘要 IV
List of figures VIII
List of tables XIV
Abbreviations and nomenclature XV

Chapter 1: Introduction 1
1.1 MEMS and microfluidic technology 1
1.2 Cell fusion 2
1.2.1 Virus-mediated cell fusion 3
1.2.2 PEG (Polyethylene glycol)-based fusion 4
1.2.3 Electrofusion 5
1.3 Cell fusion on microfluidic devices 7
1.4 Optically-induced cell fusion system 10
1.5 Motivation and objectives 13





Chapter 2: Materials and methods 20
2.1 Design of the optically-induced cell fusion (OICF) chip 20
2.2 Design of microstructures for cell pairing 20
2.3 Fabrication of the OICF chip 21
2.3.1 Lithography process 21
2.3.2 Chip assembly 22
2.4 Sample preparation 23
2.4.1 Cells 23
2.4.2 OICF chip 23
2.4.3 PEG-based fusion on chip 24
2.5 Experimental procedure of OICF 24
2.6 Experimental setup 25
2.7 Numerical simulation of local electric field 26

Chapter 3: Results and discussion 33
3.1 Numerical simulation of OICF 33
3.2 Cell pairing in microstructures 34
3.3 Optically-induced cell fusion 35
3.4 PEG fusion on cell pairing structures 36

Chapter 4: Conclusions and future perspectives 44
4.1 Conclusions 44
4.2 Future perspectives 45

References 46

[1] J. C. McDonald, and G. M. Whitesides, “Poly (dimethylsiloxane) as a material for fabricating microfluidic devices,” Accounts of chemical research, 2002, 35(7): pp.491-499.
[2] E. H. Tay, “Microfluidics and BioMEMS applications,” Norwell, MA, USA: Kluwer Academic Publishers, 2002.
[3] G. Pontecorvo, “Production of mammalian somatic cell hybrids by means of polyethylene glycol treatment,” Somatic cell genetics, 1975, 1(4): pp.397-400.
[4] N. Hu, J. Yang, Z. Q. Yin, Y. Ai, S. Qian, I. B. Svir, B. Xia, J. W. Yan, W. S. Hou, and X. L. Zheng, “A high‐throughput dielectrophoresis‐based cell electrofusion microfluidic device,” Electrophoresis, 2011, 32(18): pp.2488-2495.
[5] C. Wild, T. Greenwell, T. Matthews, “A synthetic peptide from HIV-1 gp41 is a potent inhibitor of virus-mediated cell—cell fusion,” AIDS research and human retroviruses, 1993, 9(11): pp.1051-1053.
[6] M. Tada, T. Tada, L. Lefebvre, S. C. Barton and M. A. Surani, “Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells,” The EMBO journal, 1997, 16(21): pp.6510-6520.


[7] V. L. Sukhorukov, R. Reuss, J. M. Endter, S. Fehrmann, A.K. Globa, P. Geßner, A. Steinbach, K. J. Müller, A. Karpas, U. Zimmermann, H. Zimmermann, “A biophysical approach to the optimisation of dendritic-tumour cell electrofusion,” Biochemical and biophysical research communications, 2006, 346(3): pp.829-839.
[8] G. Vassilopoulos, P. R. Wang, and D. W. Russell, “Transplanted bone marrow regenerates liver by cell fusion,” Nature, 2003, 422(6934): pp.901-904.
[9] Y. Okada, “Analysis of giant polynuclear cell formation caused by HVJ virus from Ehrlich's ascites tumor cells: I. Microscopic observation of giant polynuclear cell formation,” Experimental cell research, 1962, 26(1): pp.98-107.
[10] S. Knutton, “The mechanism of virus-induced cell fusion,” Micron, 1978, 9(3): pp.133-154.
[11] K. N. Kao and M. R. Michayluk, “A method for high-frequency intergeneric fusion of plant protoplasts,” Planta, 1974, 115(4): pp.355-367.
[12] G. Pontecorvo, “Production of mammalian somatic cell hybrids by means of polyethylene glycol treatment,” Somatic cell genetics, 1975, 1(4): pp.397-400.
[13] B. R. Lentz, “Polymer-induced membrane fusion: potential mechanism and relation to cell fusion events,” Chemistry and physics of lipids, 1994, 73(1): pp.91-106.

[14] B. R. Lentz, “PEG as a tool to gain insight into membrane fusion,” European Biophysics Journal, 2007, 36(4): pp.315-326.
[15] S. Knutton, “Studies of membrane fusion. III. Fusion of erythrocytes with polyethylene glycol,” Journal of cell science, 1979, 36(1): pp.61-72.
[16] U. Zimmermann, G. Pilwat, and F. Riemann, “Dielectric breakdown of cell membranes,” Membrane transport in plants, Springer Berlin Heidelberg, 1974: pp.146-153.
[17] H. P. Schwan, “Biophysics of the interaction of electromagnetic energy with cells and membranes,” Biological effects and dosimetry of nonionizing radiation, Springer US, 1983, 49: pp.213-231.
[18] I. P. Sugar, F. Walter, and N. Eberhard, “Model of cell electrofusion: membrane electroporation, pore coalescence and percolation,” Biophysical chemistry, 1987, 26(2): pp.321-335.
[19] T. Y. Tsong, “Electroporation of cell membranes,” Biophysical journal, 1991, 60(2): pp.297-306.
[20] N. Hu, J. Yang, Z. Q. Yin, Y. Ai, S. Qian, I. B. Svir, B. Xia, J. W. Yan, W. S. Hou, and X. L. Zheng, “high‐throughput dielectrophoresis‐based cell electrofusion microfluidic device,” Electrophoresis, 2011, 32(18): pp.2488-2495.
[21] M. Gel, Y. Kimura, O. Kurosawa, H. Oana, H. Kotera, M. Washizu, “Dielectrophoretic cell trapping and parallel one-to-one fusion based on field constriction created by a micro-orifice array,” Biomicrofluidics, 2010, 4(2): pp.022808.
[22] A. M. Skelley, O. Kirak, H. Suh, R. Jaenisch, J. Voldman, “Microfluidic control of cell pairing and fusion,” Nature methods, 2009, 6(2): pp.147-152.
[23] B. Dura, Y. Liu, and J. Voldman, “Deformability-based microfluidic cell pairing and fusion,” Lab on a Chip, 2014, 14(2): pp.2783-2790.
[24] P. Y. Chiou, A. T. Ohta, and M. C. Wu, “Massively parallel manipulation of single cells and microparticles using optical images,” Nature, 2005, 436(7049): pp.370-372.
[25] S. J. Williams, A. Kumar, N. G. Green and S. T. Wereley, “A simple, optically induced electrokinetic method to concentrate and pattern nanoparticles,” Nanoscale,” 2009, 1(1): pp.133-137.
[26] M. W. Lee, Y. H. Lin, and G. B. Lee, “Manipulation and patterning of carbon nanotubes utilizing optically induced dielectrophoretic forces,” Microfluidics and Nanofluidics, 2010, 8(5): pp.609-617.
[27] R. Schwarz, F. Wang, and M. Reissner, “Fermi level dependence of the ambipolar diffusion length in amorphous silicon thin film transistors,” Applied physics letters, 1993, 63(8): pp.1083-1085.
[28] A. Ashkin, “Acceleration and trapping of particles by radiation pressure,” Physical review letters, 1970, 24(4): pp.156.
[29] Y. Liu, G. J. Sonek, M. W. Berns, and B. J. Tromberg, “Physiological monitoring of optically trapped cells: assessing the effects of confinement by 1064-nm laser tweezers using microfluorometry,” Biophysical journal, 1996, 71(4): pp.2158-2167.
[30] Y. H. Lin, and G. B. Lee, “Optically induced flow cytometry for continuous microparticle counting and sorting,” Biosensors and Bioelectronics, 2008, 24(4): pp.572-578.
[31] C. H. Wang, Y. H. Lee, H. T. Kuo, W. F. Liang, W. J Li, and G. B. Lee, “Dielectrophoretically-assisted electroporation using light-activated virtual microelectrodes for multiple DNA transfection,” Lab on a Chip, 2014, 14(3): pp.592-601.
[32] S. L. Neale, A. T. Ohta, H. Y. Hsu, J. K. Valley, A. Jamshidi, and M. C. Wu, “Trap profiles of projector based optoelectronic tweezers (OET) with HeLa cells,” Optics Express, 2009, 17(7): pp.5231-5239.
[33] Y. Zhao, X. T Zhao, D. Y. Chen, Y. N. Luo, M. Jiang, C. Wei, R. Long, W. T. Yue, and J. B. Wang, “Tumor cell characterization and classification based on cellular specific membrane capacitance and cytoplasm conductivity,” Biosensors and Bioelectronics, 2014, 57: pp.245-253.

[34] X. Yu, P. A. McGraw, F. S. House, and J. E. Crowe Jr, “An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies,” Journal of Immunological Methods, 2008, 336(2): pp.142-151.
[35] V. L. Sukhorukov, R. Reuss, J. M. Endter, S. Fehrmann, A. Katsen-Globa, P. Geßner, A.Steinbach, K. J. Müller, A. Karpas, U. Zimmermann, H. Zimmermann, "A biophysical approach to the optimisation of dendritic-tumour cell electrofusion,” Biochemical and Biophysical Research Communications, 2006, 346(3): pp.829-839.