細胞融合是現今重要的生物應用，其範圍涵括多種生醫應用如誘導幹細胞的製造，癌症免疫治療，基因定位，單一複製抗體製造和組織再生。現今主要有三種方式可以實現細胞融合，大致分為物理性，化學性和生物性的方法。然而，如何同時精準地配對細胞和維持穩定的細胞接觸是限制細胞融合達到更高成功率和產量的兩大主因。基於以上種種原因，我們在這項研究中提出了一種新的概念和方法。此方法主要是使用光誘發的介電泳和區域化電場在無結構的的微流體晶片中完成自動化的細胞配對以及細胞融合。我們將由光圖形誘發所產生的「虛擬」電極視作如真實的電極和夾具一般，幫助我們配對細胞以及施加電場進行細胞的電穿孔藉此觸發細胞融合。在經過光介電泳配對細胞後，由系統的虛擬電極搭配特定的光圖形所產生的區域化電場可以在兩個細胞接觸的區域誘發相對最大的膜電位，因此細胞融合得以開始進行。這項研究成功證實了在無微結構輔助下單獨只用光圖形觸發細胞融合的可行性. 我們的系統在胰臟癌細胞和肺腺癌細胞的融合上達到了最高9.67%的成功率這也表示這項技術在不久的將來非常有機會成功優化並應用在大規模的細胞融合。

Cell fusion is a vital technology, which has been well explored for various biomedical applications including production of induced stem cells, cancer immunotherapy, gene mapping, monoclonal antibody production and tissue regeneration. There are three major approaches to realize cell fusion, including physical, chemical and biological methods. However, none of them can perfectly solve two main limitations at the same time, cell pairing and cell contact, to achieve high efficiency and yields. Hence, in this study, we reported a new method using two techniques, including optically-induced dielectrophoresis (ODEP) and optically-induced locally-enhanced electric field (OILEF) to accomplish accurate cell pairing and cell fusion automatically on a structure-free device. “Virtual” electrodes induced by light patterns were served as manipulators and electrodes for cell pairing and cell fusion. After pairing cells by using ODEP techniques, locally-enhanced electric field generated by virtual electrodes could induce a maximum transmembrane potential at the cell contact area such that cell fusion could be triggered. This study successfully demonstrated the feasibility of this new approach that realized cell fusion by using simple light pattern only without any microstructure. A fusion yield of 9.67% was achieved in the cell pair of Pan 1 and A549 cells. It suggested that this promising technique could automatically conduct massive cell fusion within a group of cells in the near future.

Abstract I
摘要 II
Table of contents III
List of figures VI
List of tables XI
Abbreviations and nomenclature XIII
Chapter 1: Introduction 1
1.1 MEMS and microfluidic technology 1
1.2 Background and literature survey 2
1.2.1 Cell fusion 2
1.2.2 Cell fusion on microfluidic devices 2
1.2.3 Amorphous silicon as a photoconductive material 3
1.2.4 ODEP for particle manipulation 4
1.2.5 Critical transmembrane potential for electrofusion 7
1.2.6 Optically-induced cell fusion system 7
1.3 Motivation and novelty 9
Chapter 2: Materials and methods 10
2.1 Chip design 10
Structure-free chip 10
Structured chip 10
2.2 Fabrication process 13
2.2.1 Photolithography process of SU-8 microstructure 13
2.2.2 Chip assembly process 14
Structure-free chip 14
Structured chip 14
2.3 Sample preparation 17
2.3.1 Cells 17
2.3.2 OICF chip 17
2.4 Cell manipulation 18
2.5 Cell electroporation 18
2.6 Experimental procedure 21
Structure-free OICF 21
Structured OICF 21
2.7 Numerical simulation of localized electric field 24
2.8 Experimental setup 26
2.8.1 ODEP system 26
2.8.2 Cell culture system outside typical incubator 26
Chapter 3: Results and discussion 29
3.1 Numerical simulation result of structure-free OICF 29
3.2 Characterization of OICF chip 32
3.2.1 ODEP force 32
3.2.2 Cell pairing in microstructures 33
3.3 Optically-induced cell fusion 36
3.3.1 Cell electroporation in OICF chip 36
3.3.2 Cell electrofusion 36
Chapter 4: Conclusions and future perspectives 41
4.1 Conclusions 41
4.2 Future perspectives 42
References 43

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