在全球，癌症一直是致死的主要原因。過去，免疫療法是治療特定類型癌症的 關鍵。探索更新類型的免疫療法將對未來癌症的治療方式產生影響，免疫療法 包括多種治療方式，有些使用補充劑在廣義上增強了身體的免疫系統，或是幫 助調節免疫系統以特別攻擊癌細胞。免疫系統記憶了在體內發現的所有化學物 質，一旦免疫系統識別到任何新物質都會觸發警報，促使免疫系統對其進行攻 擊。然而，免疫系統難以輕易攻擊癌細胞，這是因為癌症即是體內細胞發生突 變並開始不受控制地生長，所以癌細胞通常不被免疫系統識別為外來細胞。因 此，免疫系統自身對抗癌症的能力是有限的，導致許多免疫系統健康的人仍然 會患上癌症。有時，免疫系統不會將癌細胞識別為外來細胞，因為它們與正常 細胞的區別不夠明顯;有時，免疫系統會識別癌細胞，但反應不足以根除癌細胞。 為了解決這個問題，研究人員發現了許多增強免疫系統反應的方法，使其能夠 消除癌症細胞，在眾多方法中，細胞融合作為癌症免疫治療的方法引起了研究 人員的興趣，而微流體技術在實現融合細胞上發揮了關鍵作用。基於微流體晶 片讓研究人員能對細胞進行精確操作，從而實現細胞的一對一配對，在電極之 間施加電場來融合細胞，並能將融合的細胞取出進行更近一步的實驗。
此篇論文提出了一種能用於高流量細胞操作的微流體介電泳實驗室晶片。利用 聚二甲基矽氧烷 (PDMS)，一種不導電的聚合物作為絕緣體。利用剝離技術在 ITO 基板上形成具有大量孔洞排列的PDMS薄膜。施加交流電場後，PDMS 薄膜 會在兩個 ITO 玻璃之間產生介電泳力所需的空間不均勻性，絕緣結構在 iDEP 器 件中產生電場梯度使得非均勻場充滿整個體介質，而 ITO 的透明特性使其成為 光學量化的理想選擇。該晶片的優勢是僅經由調整三個參數進行優化以適應任 何細胞大小:PDMS 厚度、微孔直徑和電場。此種微流控晶片解決了一些缺點， 例如 (i) 晶片處理:此研究晶片在操作過程中易於處理，因為只有一個入口和一 個出口，(ii) 高吞吐量:此研究晶片可以擴展用以融合更多細胞數量 ，(iii) 複雜 性:晶片的可訪問性設計使細胞配對和融合過程更容易，(iv) 兼容性:此研究 晶片可以根據所需的細胞類型進行修改。我們建立了CT26 和 BMDC 之間的細 胞模式和融合。在精確配對的細胞中，我們實現了高達 70%(3000-3500 個細胞)
的融合效率。四天後，對融合細胞進行細胞活力調查，以評估此研究晶片是否 具有生物相容性且不受暴露電場的影響，我們觀察到了 60%(1800-2200 個細胞) 的活細胞。此外，我們從細胞生物學的角度研究了融合細胞的特徵，包括來自 CT26 和 BMDC 的組合熒光標記細胞內成分、混合細胞形態。

Globally, cancer has been the main cause of mortality. Immunotherapy has been a critical component of treating certain types of cancer over the previous few decades. Newer types of immune therapies are now being explored, and they will have an impact on how cancer is treated in the future. Immunotherapy encompasses a variety of treatment modalities. Certain supplements strengthen the body's immune system in a broad sense. Others assist in conditioning the immune system to particularly attack cancer cells. The immune system contains a record of all chemicals discovered in the body. Any novel substance recognized by the immune system triggers an alarm, prompting the immune system to attack it. However, the immune system has a more difficult time attacking cancer cells. This is because cancer begins when cells undergo mutations and begin to grow uncontrollably. Cancer cells are not usually recognized as a foreign body by the immune system. Clearly, the immune system's ability to fight cancer on its own is limited, as seen by the fact that many people with healthy immune systems nevertheless develop cancer. Occasionally, the immune system does not recognize cancer cells as foreign material because they are not sufficiently distinct from normal cells. Occasionally, the immune system recognizes cancer cells, but the response is insufficient to eradicate the tumor. To combat this, researchers have discovered ways to boost the immune system's response, allowing it to eliminate them.
Cell fusion could be an answer to this approach. Cell fusion has triggered interest among researchers in the last few decades as a promising tool for cancer immunotherapy. In recent years, microfluidics has played a key role in the development of fused cells. The microfluidics-based devices offer precise manipulation of cells, thereby achieving one-on-one cell pairing of desired cells. The cells are fused using the applied electric field between a pair of electrodes. The fused cells are collected from the chip, and a further study can be performed.
We demonstrate an insulator-based dielectrophoretic lab-chip (iDEP-LC) for highly efficient cell trapping, pairing and fusion. Polydimethylsiloxane (PDMS) has been used as an insulator. We present a lift-off approach for forming a thin PDMS layer on ITO glass with an array of holes. The presence of a PDMS layer generates non uniform electric field required to generate dielectrophoretic force between two ITO glasses. Due to transparent nature of ITO, is an excellent choice as it enables optical quantification. The proposed design can be reconfigured for any cell size by optimizing membrane thickness, diameter of microwell, and electric field. The chip addresses commonly faced problems such as (i) handling: presence of single inlet/outlet makes it easy to operate, (ii) high throughput: the chip is scalable for high fused cell number, (iii) complexity: the chip is simple in design and thus easy to use, (iv) chip compatibility: the chip can be reconfigured for different cell types.
Cell pairing and fusion among BMDC and CT26 cells has been demonstrated. The fusion efficiency of 70% (3000-3500 cells) was observed between correctly paired cells. The biocompatibility of device fabricated using lift-off process was also carried out. A study related to effect of electric field on cell viability was also carried out. A 4-day cell viability analysis revealed that 60 % (1800-2200 cells) of cells are viable. Additionally, we evaluated the biological characteristics of fused cells, including combined fluorescence-labeled intracellular components from CT26 and BMDC, mixed cell morphology.

ABSTRACT i
摘要 iii
TABLE OF CONTENTS v
LIST OF FIGURES viii
LIST OF TABLES xvii
ACKNOWLEDGEMENT xviii
ABBREVIATIONS xx
CHAPTER 1 INTRODUCTION 1
1.1 History 2
1.2 Cell fusion 2
1.2.1 Conventional electrofusion 2
1.2.2 Why the move towards microfluidics-based electrofusion devices? 3
1.3 Thesis Outline 4
1.4 Electrofusion principle 5
1.5 Cell fusion technology 7
1.5.1 Virus mediated fusion 7
1.5.2 Chemical method (PEG) 8
1.5.3 Physical method (Electrofusion) 8
1.6 Conclusion 17
CHAPTER 2 CELL ELECTROFUSION: CONDITIONS AND CHIP REQUIREMENTS 18
2.1 Buffer parameters affecting cell fusion 18
2.1.1 Cell types 18
2.1.2 Osmolality of buffer solution 19
2.1.3 Conductivity of buffer 19
2.2 Electrical parameters affecting cell fusion 19
2.2.1 Geometry of electrode 19
2.3 Chip parameters 20
2.3.1 Chip material 20
2.3.2 Electrode material and configuration 20
2.4 Fusion process visualization 21
CHAPTER 3 MICROFLUIDIC FLIP-CHIP 22
3.1 Materials and Methods 22
3.1.1 Device design 22
3.1.2 Device fabrication 23
3.1.3 PDMS membrane optimization 24
3.1.4 Modeling and simulation 25
3.1.5 Cell preparation 27
3.1.6 Experimental setup 28
3.1.7 Device operation 29
3.2 Image acquisition and analysis 31
3.3 Results 31
3.3.1 Cell pairing 31
3.3.2 Cell electrofusion 31
3.3.3 Effect of electric field on fusion efficiency 33
3.3.4 Effect of membrane thickness 34
3.3.5 Effect of washing flow rate 35
3.3.6 Characterization of fused cells 35
3.3.7 Cell viability in 96-well plate 37
3.4 Discussion 38
CHAPTER 4 iDEP CHIP: DESIGN AND FABRICATION 39
4.1 Need for iDEP chip 39
4.2 Polarization of cells in the presence of electric field 39
4.3 Dielectrophoretic force (DEP) 40
4.3.1 Transmembrane potential 42
4.4 iDEP device design 43
4.5 Numerical simulations 44
4.5.1 FEM Simulation using COMSOL Multiphysics 44
4.5.2 Analysis of transmembrane potential 47
4.6 Device fabrication 50
CHAPTER 5 MATERIALS AND METHODS 52
5.1 Cell culture medium preparation 52
5.1.1 Medium for CT26 cells 52
5.1.2 Medium for Bone marrow derived dendritic cells (BMDC) 52
5.2 DEP buffer protocol 53
5.3 Cell culture and preparation 54
5.3.1 Bone marrow derived dendritic cells (BMDC) 54
5.3.2 CT26 cells 54
5.4 Pretreatment of microfluidic chip 55
5.5 Cell preparation for DEP manipulation 56
5.6 Experimental setup 56
5.7 Device operation 57
5.7.1 Cell pairing 57
5.7.2 Cell fusion 58
5.8 PrestoBlue Assay 59
CHAPTER 6 RESULTS AND DISCUSSION 60
6.1 PDMS membrane optimization 60
6.2 Step-by-step process of cell electrofusion 61
6.2.1 Cell trapping 61
6.2.2 Cell pairing 63
6.2.3 Cell electrofusion 64
6.3 Time-series imaging of cell-electrofusion process 66
6.4 Effect of flow rate on trapping efficiencies 67
6.4.1 Flow rate optimization for CT26 67
6.4.2 Effect of washing flow rate on CT26 cells 68
6.4.3 Flow rate optimization for BMDC 69
6.4.4 Effect of washing flow rate on BMDC 69
6.5 Effect of Electric field on fusion efficiency 70
6.6 Characterization of fused cells 71
6.7 Cell viability using PrestoBlue assay 73
CHAPTER 7 CONCLUSION AND FUTURE SCOPE 75
7.1 Future scope 77
7.1.1 A step closer to standardization of microfluidic chip: Use of glass microfluidics 77
7.1.2 Single cell encapsulation 78
LIST OF PUBLICATIONS 80
BIBLIOGRAPHY 83

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