常規的細胞培養方法主要以二維細胞培養模式開發。儘管它們在生物醫學研究中具有重要價值，但它們無法支持許多細胞類型的組織特異性分化功能，也無法準確預測體內組織功能和藥物活性。這些局限性導致人們對三維細胞培養模型的興趣日益濃厚，而三維細胞培養模型可以更好地代表生物組織的空間和化學複雜性。現今已經有許多三維器官與腫瘤模型被開發，因為它們可以較好的模擬真實人體環境，與二維培養相比可以得到更確實的藥物反應。3D培養方法可以分為以下幾種類別：（i）吊降方法； （ii）非粘性表面方法； （iii）懸浮培養；（iv）基於支架的水凝膠； （v）磁懸浮和生物印刷； （vi）微流體方法，各自有許多的優點以及缺點。
我們的研究設計了一種微流體懸浮液滴式芯片，由三層PMMA板組成的結構，能夠產生高通量的三維腫瘤球體用於長期培養，並且可以簡單地進行液體交換，注入藥物或奈米粒子甚至另一種細胞，並讓我們可以詳細地了解三維腫瘤微觀結構、藥物的效果、奈米粒子載體與腫瘤球體的相關作用或是滲透程度的觀察、甚至是與免疫細胞的相互作用。這些三維腫瘤模型芯片提供了一個概念驗證平台，可以用於篩選抗癌藥物，識別顆粒滲透效果和進行免疫治療。
關鍵字: 3D細胞培養模型， 高通量， 藥物篩選， 粒子滲透，免疫治療

Conventional cell culture methods are mainly developed in a two-dimensional cell culture model. Although they have important value in biomedical research, they cannot support the tissue-specific differentiation function of many cell types, nor can they accurately predict tissue function and drug activity in the body. These limitations have led to increasing interest in three-dimensional cell culture models, which can better represent the spatial and chemical complexity of biological tissues. Nowadays, many three-dimensional organ and tumor models have been developed, because they can better simulate the real human environment, and can get more reliable drug response than two-dimensional culture. 3D culture methods can be divided into the following categories: (i) suspension method; (ii) non-stick surface method; (iii) suspension culture; (iv) scaffold-based: hydrogel; (v) magnetic suspension and bioprinting ; (Vi) Microfluidic methods each have many advantages and disadvantages.
Our research designed a microfluidic suspension droplet chip with a three-layer PMMA plate structure that can produce high-throughput three-dimensional tumor spheroids for long-term culture and can simply exchange liquids and inject drugs or nanometers. Particles or even another type of cell, and allow us to understand in detail the three-dimensional tumor microstructure, the effects of drugs, the correlation between nanoparticle carriers and tumor spheroids, or the observation of the degree of penetration, and even the interaction with immune cells. These three-dimensional tumor model chips provide a proof-of-concept platform that can be used to screen anti-cancer drugs, identify particle penetration effects, and perform immunotherapy.
Keywords— 3D cell culture model; high-throughput; drug screening; particle penetration; immunotherapy

中文摘要 I
Abstract II
致謝 IV
Chapter 1 Introduction 1
Chapter 2 Literature review and theory 3
2.1 3D cell cultures 3
2.1.1 Comparison of 3D cell cultures and 2D cell cultures 4
2.1.2 Cell cultures as a research model 6
2.2 Introduction of tumor on a chip 7
2.2.1 The tumor niche 8
2.2.2 Tumor on chip is superior alternative for emulating the tumor micro-niche 10
2.2.3 Fabrication techniques of tumor spheroids 14
2.3 Different models of 3D culture of tumor chip 17
2.3.1 Hanging drop methods 19
2.3.2 Spontaneous Spheroid Formation: Non-Adherent Surface Methods/Ultra Low Attachment Plates 20
2.3.3 Suspension Culture 20
2.3.4 Scaffold-Based Models: Hydrogels 21
2.3.5 Magnetic Levitation 21
2.3.6 High-throughput microfluidic hanging drop chip 22
Chapter 3 Experimental section 24
3.1 Materials 24
3.2 Apparatus 26
3.3 Method 27
3.3.1 Fabrication and design of chip 27
3.3.2 Cell culture 28
3.3.3 Tumor spheroids formation 28
3.3.4 Tumor spheroids fluorescent staining 29
3.3.5 Drug Screening test 29
3.3.6 Penetration of the nanoparticles 30
3.3.7 Infusion of immune cells 32
Chapter 4 Results and Discussions 33
4.1 Characterization of chip 33
4.2 Tumor spheroids formation 34
4.3 Precision Medicine and Drug Screening model for HM4 Tumor Spheroids 38
4.4 Penetration of Fe3O4-Den-Exos into tumor spheroids 41
4.5 Immunotherapy model 45
Chapter 5 Conclusions 48
Reference 49

1 Kapałczyńska, M. et al. 2D and 3D cell cultures–a comparison of different types of cancer cell cultures. Archives of medical science: AMS 14, 910 (2018).
2 Ryan, S.-L. et al. Drug discovery approaches utilizing three-dimensional cell culture. Assay and drug development technologies 14, 19-28 (2016).
3 Pampaloni, F., Reynaud, E. G. & Stelzer, E. H. The third dimension bridges the gap between cell culture and live tissue. Nature reviews Molecular cell biology 8, 839-845 (2007).
4 Nelson, C. M. & Bissell, M. J. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Annu. Rev. Cell Dev. Biol. 22, 287-309 (2006).
5 Hamburger, A. W. & Salmon, S. E. Primary bioassay of human tumor stem cells. Science 197, 461-463 (1977).
6 Mazzoleni, G., Di Lorenzo, D. & Steimberg, N. Modelling tissues in 3D: the next future of pharmaco-toxicology and food research? Genes & nutrition 4, 13 (2009).
7 Griffith, L. G. & Swartz, M. A. Capturing complex 3D tissue physiology in vitro. Nature reviews Molecular cell biology 7, 211-224 (2006).
8 Lee, J., Cuddihy, M. J. & Kotov, N. A. Three-dimensional cell culture matrices: state of the art. Tissue Engineering Part B: Reviews 14, 61-86 (2008).
9 Benya, P. D. & Shaffer, J. D. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 30, 215-224 (1982).
10 Bray, F. et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians 68, 394-424 (2018).
11 Trujillo-de Santiago, G. et al. The Tumor-on-Chip: Recent Advances in the Development of Microfluidic Systems to Recapitulate the Physiology of Solid Tumors. Materials 12, 2945 (2019).
12 Kim, J.-Y. et al. 3D spherical microtissues and microfluidic technology for multi-tissue experiments and analysis. Journal of biotechnology 205, 24-35 (2015).
13 Han, B., Qu, C., Park, K., Konieczny, S. F. & Korc, M. Recapitulation of complex transport and action of drugs at the tumor microenvironment using tumor-microenvironment-on-chip. Cancer letters 380, 319-329 (2016).
14 Erdogan, B. et al. Cancer-associated fibroblasts promote directional cancer cell migration by aligning fibronectin. Journal of Cell Biology 216, 3799-3816 (2017).
15 Gilkes, D. M., Semenza, G. L. & Wirtz, D. Hypoxia and the extracellular matrix: drivers of tumour metastasis. Nature Reviews Cancer 14, 430-439 (2014).
16 Soltani, M. & Chen, P. Effect of tumor shape and size on drug delivery to solid tumors. Journal of biological engineering 6, 4 (2012).
17 Heylman, C., Sobrino, A., Shirure, V. S., Hughes, C. C. & George, S. C. A strategy for integrating essential three-dimensional microphysiological systems of human organs for realistic anticancer drug screening. Experimental biology and medicine 239, 1240-1254 (2014).
18 Gengenbacher, N., Singhal, M. & Augustin, H. G. Preclinical mouse solid tumour models: status quo, challenges and perspectives. Nature Reviews Cancer 17, 751-765 (2017).
19 Joffe, A. R., Bara, M., Anton, N. & Nobis, N. The ethics of animal research: a survey of the public and scientists in North America. BMC medical ethics 17, 17 (2016).
20 Katt, M. E., Placone, A. L., Wong, A. D., Xu, Z. S. & Searson, P. C. In vitro tumor models: advantages, disadvantages, variables, and selecting the right platform. Frontiers in bioengineering and biotechnology 4, 12 (2016).
21 Caballero, D. et al. Organ-on-chip models of cancer metastasis for future personalized medicine: From chip to the patient. Biomaterials 149, 98-115 (2017).
22 Sylvester, D., M Hattersley, S., D Stafford, N., J Haswell, S. & Greenman, J. Development of microfluidic-based analytical methodology for studying the effects of chemotherapy agents on cancer tissue. Current Analytical Chemistry 9, 2-8 (2013).
23 Wang, S. et al. Study on invadopodia formation for lung carcinoma invasion with a microfluidic 3D culture device. PLoS One 8, e56448 (2013).
24 Nath, S. & Devi, G. R. Three-dimensional culture systems in cancer research: Focus on tumor spheroid model. Pharmacology & therapeutics 163, 94-108 (2016).
25 Costa, E. C. et al. 3D tumor spheroids: an overview on the tools and techniques used for their analysis. Biotechnology advances 34, 1427-1441 (2016).
26 Friedrich, J., Seidel, C., Ebner, R. & Kunz-Schughart, L. A. Spheroid-based drug screen: considerations and practical approach. Nature protocols 4, 309 (2009).
27 Sant, S. & Johnston, P. A. The production of 3D tumor spheroids for cancer drug discovery. Drug Discovery Today: Technologies 23, 27-36 (2017).
28 Lazzari, G. et al. Multicellular spheroid based on a triple co-culture: A novel 3D model to mimic pancreatic tumor complexity. Acta biomaterialia 78, 296-307 (2018).
29 Nunes, A. S., Barros, A. S., Costa, E. C., Moreira, A. F. & Correia, I. J. 3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs. Biotechnology and bioengineering 116, 206-226 (2019).
30 Lin, R.-Z., Chou, L.-F., Chien, C.-C. M. & Chang, H.-Y. Dynamic analysis of hepatoma spheroid formation: roles of E-cadherin and β1-integrin. Cell and tissue research 324, 411-422 (2006).
31 Kelm, J. M., Timmins, N. E., Brown, C. J., Fussenegger, M. & Nielsen, L. K. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnology and bioengineering 83, 173-180 (2003).
32 Lee, W. G., Ortmann, D., Hancock, M. J., Bae, H. & Khademhosseini, A. A hollow sphere soft lithography approach for long-term hanging drop methods. Tissue Engineering Part C: Methods 16, 249-259 (2010).
33 Frey, O., Misun, P. M., Fluri, D. A., Hengstler, J. G. & Hierlemann, A. Reconfigurable microfluidic hanging drop network for multi-tissue interaction and analysis. Nature communications 5, 1-11 (2014).
34 Hoarau-Véchot, J., Rafii, A., Touboul, C. & Pasquier, J. Halfway between 2D and animal models: are 3D cultures the ideal tool to study cancer-microenvironment interactions? International journal of molecular sciences 19, 181 (2018).
35 Jørgensen, A. et al. Hanging drop cultures of human testis and testis cancer samples: a model used to investigate activin treatment effects in a preserved niche. British journal of cancer 110, 2604-2614 (2014).
36 Kuo, C.-T. et al. Three-dimensional spheroid culture targeting versatile tissue bioassays using a PDMS-based hanging drop array. Scientific reports 7, 1-10 (2017).
37 Sutherland, R. M., Inch, W. R., McCredie, J. A. & Kruuv, J. A multi-component radiation survival curve using an in vitro tumour model. International Journal of Radiation Biology and Related Studies in Physics, Chemistry and Medicine 18, 491-495 (1970).
38 Lin, R. Z. & Chang, H. Y. Recent advances in three‐dimensional multicellular spheroid culture for biomedical research. Biotechnology Journal: Healthcare Nutrition Technology 3, 1172-1184 (2008).
39 Fang, Y. & Eglen, R. M. Three-dimensional cell cultures in drug discovery and development. Slas discovery: Advancing Life Sciences R&D 22, 456-472 (2017).
40 Souza, G. R. et al. Three-dimensional tissue culture based on magnetic cell levitation. Nature nanotechnology 5, 291-296 (2010).
41 Wu, H.-W., Yao, W.-Y., Hsiao, Y.-H. & Hsu, C.-H. in 2017 IEEE 12th International Conference on Nano/Micro Engineered and Molecular Systems (NEMS). 351-354 (IEEE).