||Development of a highly reproducible microwell array platform for high-throughput tumor spheroid culture and drug assessment
||Department of BioMedical Engineering
Three-dimensional (3D) tumor culture
multicellular tumor spheroids (MCTS)
in vitro tumor model
high-throughput drug screening
三維（Three-dimensional, 3D）多細胞腫瘤球體（Multicellular spheroids, MCTSs）具備原發腫瘤微環境在生理上相關的病理特徵，近年來已成為癌症研究重要的體外模型。在過去十年中，為快速製造MCTSs，廣泛採用一種簡易的方法－雷射燒蝕U型微孔。然而，過去的研究對於微孔均勻性和陣列結構應用以及藥物測試的區分仍值得商榷。在此研究中，我們提出了一種雷射燒蝕微孔陣列的技術，該技術不僅可以實現具有相同大小的MCTSs，並且能在原位進行高通量藥物評估。研究中包含三個關鍵的雷射燒蝕參數：頻率（1-20 kHz）、工作週期（10-90％）和脈衝次數（60-700），這些參數可靈活地產生尺寸為170 至 400 µm的微孔。可以通過精確控制460 µm的水平間距（dx）和200 µm的垂直間距（dy）使260 µm大小的微孔排列成緊密陣列。在每個微孔有50、100和150個細胞等種植密度條件下，從微孔陣列中收穫的T24，A549和Huh-7 MCTSs的直徑對應於約為75至140 µm。順鉑抗癌藥物篩選驗證了二維和MCTS條件下，IC50值分別為3.5 vs. 9.1 µM（T24），11.8 vs. 277.7 µM（A549）和33.5 vs. 52.8 µM（Huh-7），並且通透性範圍由0.042至0.58 µm / min。研究結果表明：MCTS中存在較強的抗藥性，但滲透率與藥物療效無關。預期本研究提出之方法能用於簡便且高度一致的微孔製造，並可靠地生產MCTS與建立用於藥物評估的深入指南。
Three-dimensional (3D) multicellular tumor spheroids (MCTSs) have recently emerged as a landmark for cancer research due to their inherent traits physiologically relevant to primary tumor microenvironments. In the past decade, to rapidly engineer MCTSs, a facile approach – laser-ablated micro U-wells – is widely adopted. However, differentiations of both the microwell uniformities and the construction of arrays as well as drug testings resulted from above studies remain elusive. Here, we propose an improved laser-ablated microwell array technique that can not only achieve arrayed MCTSs with identical sizes but also perform high-throughput drug assessments in situ. Three critical laser ablation parameters, including Frequency (1-20 kHz), Duty Cycle (10-90%), and Pulse Number (60-700), were investigated that generated microwells flexibly with a range from 170 – 400 µm. The choice of 260 µm microwells could be optimally arranged into an array via precise control of horizontal spacing (dx) at 460 µm and vertical spacing (dy) at 200 µm amenable of cell-loss-free culture during cell seeding. Harvested T24, A549 and Huh-7 MCTSs from the microwell array corresponded to around 75 to 140 µm in diameter under seeding densities of 50, 100 and 150 cells/microwell. Anticancer drug screening of cisplatin validated the IC50 values in 2D and MCTS conditions were 3.5 vs. 9.1 µM (T24), 11.8 vs. 277.7 µM (A549) and 33.5 vs. 52.8 µM (Huh-7), and the permeability was measured from 0.067 to 0.322 µm min-1. Our findings suggest that an enhanced drug resistance were present in MCTS, yet the permeability was irrespective of the drug efficacy. The current approach is envisioned as an in-depth protocol guide for a facile and highly consistent microwell fabrication to reliably produce MCTS and establish a workflow for drug evaluation.
List of Tables VIII
List of Figures IX
List of Abbreviations XII
Chapter 1 Introduction 1
1.1 Background 1
1.2 Tumor microenvironment (TME) 2
1.3 Multicellular tumor spheroids (MCTSs) 3
1.4 Anticancer drug penetration and screening 4
1.5 CO2 laser advantage 6
1.5.1 Laser micromachining 6
1.5.2 CO2 laser ablated microwell 7
1.6 Aims of the research 8
Chapter 2 Materials and Methods 10
2.1 Experimental work flows 10
2.2 Microwell 12
2.2.1 Microwell fabrication 12
2.2.2 Microwell characterization 14
2.2.3 Microwell arrangement 14
2.3 Scanning electron microscopy (SEM) 15
2.4 Cell culture 15
2.5 Cell enumeration 15
2.6 Formation of MCTSs 16
2.7 MCTS viability assessment 18
2.8 Imaging and quantification 18
2.9 Drug screening 18
2.10 Permeability measurements 20
2.11 Statistical analysis 22
Chapter 3 Results and Discussion 23
3.1 Characterization of microwells 23
3.1.1 Effect of number of pulses 23
3.1.2 Combinations of duty cycle and number of pulses 25
3.1.3 Effect of frequency 27
3.2 Characterization of MCTS 30
3.2.1 Optimization of microwell arrangement and the morphology of MCTSs 30
3.2.2 Formation of various MCTS with different seeding density 34
3.2.3 2D and 3D MCTS anticancer drug screening 37
3.2.4 Diffusional permeability coefficients of MCTS 39
Chapter 4 Conclusions 43
Chapter 5 Future works 44
 R. L. Siegel, K. D. Miller, and A. Jemal, “Cancer statistics, 2019,” CA. Cancer J. Clin., vol. 69, no. 1, pp. 7–34, 2019.
 D. M. Gilkes, G. L. Semenza, and D. Wirtz, “Hypoxia and the extracellular matrix: Drivers of tumour metastasis,” Nature Reviews Cancer. 2014.
 M. W. Pickup, J. K. Mouw, and V. M. Weaver, “The extracellular matrix modulates the hallmarks of cancer.,” EMBO Rep., vol. 15, no. 12, pp. 1243–53, Dec. 2014.
 S. Azzi, J. K. Hebda, and J. Gavard, “Vascular permeability and drug delivery in cancers,” Front. Oncol., vol. 3, p. 211, 2013.
 L.-B. Weiswald, D. Bellet, and V. Dangles-Marie, “Spherical Cancer Models in Tumor Biology.,” Neoplasia, vol. 17, no. 1, pp. 1–15, 2015.
 G. Hamilton, “Multicellular spheroids as an in vitro tumor model,” in Cancer Letters, 1998, vol. 131, no. 1, pp. 29–34.
 S. Selimović, F. Piraino, H. Bae, M. Rasponi, A. Redaelli, and A. Khademhosseini, “Microfabricated polyester conical microwells for cell culture applications.,” Lab Chip, vol. 11, no. 14, pp. 2325–2332, 2011.
 T. Y. Tu et al., “Rapid prototyping of concave microwells for the formation of 3D multicellular cancer aggregates for drug screening,” Adv. Healthc. Mater., vol. 3, no. 4, pp. 609–616, 2014.
 J. L. Albritton et al., “Ultrahigh-throughput generation and characterization of cellular aggregates in laser-ablated microwells of poly(dimethylsiloxane),” RSC Adv., vol. 6, no. 11, pp. 8980–8991, 2016.
 C. Y. Chiu et al., “Simple in-house fabrication of microwells for generating uniform hepatic multicellular cancer aggregates and discovering novel therapeutics,” Materials (Basel)., vol. 12, no. 20, p. 3308, 2019.
 B. L. Khoo, G. Grenci, Y. B. Lim, S. C. Lee, J. Han, and C. T. Lim, “Expansion of patient-derived circulating tumor cells from liquid biopsies using a CTC microfluidic culture device,” Nat. Protoc., vol. 13, no. 1, pp. 34–58, 2018.
 B. L. Khoo et al., “Short-term expansion of breast circulating cancer cells predicts response to anti-cancer therapy,” Oncotarget, vol. 6, no. 17, pp. 15578–15593, 2015.
 S. Sant and P. A. Johnston, “The production of 3D tumor spheroids for cancer drug discovery,” Drug Discov. Today Technol., vol. 23, pp. 27–36, 2017.
 A. Albini and M. B. Sporn, “The tumour microenvironment as a target for chemoprevention,” Nature Reviews Cancer. 2007.
 M. Wang et al., “Role of tumor microenvironment in tumorigenesis,” Journal of Cancer. 2017.
 R. Z. Lin and H. Y. Chang, “Recent advances in three-dimensional multicellular spheroid culture for biomedical research,” Biotechnology Journal, vol. 3, no. 9–10. pp. 1172–1184, 2008.
 L. A. Kunz-Schughart, J. P. Freyer, F. Hofstaedter, and R. Ebner, “The Use of 3-D Cultures for High-Throughput Screening: The Multicellular Spheroid Model,” J. Biomol. Screen., vol. 9, no. 4, pp. 273–285, 2004.
 S. Däster et al., “Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment,” Oncotarget, 2017.
 J. P. Freyei and R. M. Sutherland, “Regulation of Growth Saturation and Development of Necrosisin EMT6/R0 Multicellular Spheroids by the Glucose and Oxygen Supplyl,” Cancer Res., 1986.
 W. Mueller-Klieser, J. P. Freyer, and R. M. Sutherland, “Influence of glucose and oxygen supply conditions on the oxygenation of multicellular spheroids,” Br. J. Cancer, 1986.
 D. Khaitan, S. Chandna, M. B. Arya, and B. S. Dwarakanath, “Establishment and characterization of multicellular spheroids from a human glioma cell line; implications for tumor therapy,” J. Transl. Med., 2006.
 B. Desoize and J. C. Jardillier, “Multicellular resistance: A paradigm for clinical resistance?,” Critical Reviews in Oncology/Hematology. 2000.
 Shreya Raghavan, “Comparative analysis of tumor spheroid generation techniques for differential in vitro drug toxicity,” Oncotarget, 2016.
 F. Hirschhaeuser, H. Menne, C. Dittfeld, J. West, W. Mueller-Klieser, and L. A. Kunz-Schughart, “Multicellular tumor spheroids: An underestimated tool is catching up again,” J. Biotechnol., vol. 148, no. 1, pp. 3–15, 2010.
 C. R. Thoma, M. Zimmermann, I. Agarkova, J. M. Kelm, and W. Krek, “3D cell culture systems modeling tumor growth determinants in cancer target discovery,” Adv. Drug Deliv. Rev., vol. 69–70, pp. 29–41, 2014.
 E. C. Costa, V. M. Gaspar, P. Coutinho, and I. J. Correia, “Optimization of liquid overlay technique to formulate heterogenic 3D co-cultures models,” Biotechnol. Bioeng., 2014.
 J. M. Yuhas, A. P. Li, A. O. Martinez, and A. J. Ladman, “A Simplified Method for Production and Growth of Multicellular Tumor Spheroids,” Cancer Res., 1977.
 M. Zanoni et al., “3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained.,” Sci. Rep., vol. 6, no. August 2015, p. 19103, 2016.
 G. H. G. H. G.-H. Lee et al., “Networked concave microwell arrays for constructing 3D cell spheroids,” Biofabrication, vol. 10, no. 1, p. 015001, Nov. 2018.
 G. H. Lee, J. S. Lee, X. Wang, and S. Hoon Lee, “Bottom-Up Engineering of Well-Defined 3D Microtissues Using Microplatforms and Biomedical Applications,” Adv. Healthc. Mater., vol. 5, no. 1, pp. 56–74, Jan. 2016.
 V. Lavanya, M. Adil, N. Ahmed, A. K. Rishi, and S. Jamal, “Small molecule inhibitors as emerging cancer therapeutics,” Integr Cancer Sci Ther., 2014.
 K. Imai and A. Takaoka, “Comparing antibody and small-molecule therapies for cancer,” Nature Reviews Cancer. 2006.
 S. Zhong, J.-H. Jeong, Z. Chen, Z. Chen, and J.-L. Luo, “Targeting Tumor Microenvironment by Small-Molecule Inhibitors,” Transl. Oncol., vol. 13, no. 1, pp. 57–69, 2020.
 A. I. Minchinton and I. F. Tannock, “Drug penetration in solid tumours,” Nature Reviews Cancer. 2006.
 G. Mehta, A. Y. Hsiao, M. Ingram, G. D. Luker, and S. Takayama, “Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy,” J. Control. Release, vol. 164, no. 2, pp. 192–204, 2012.
 P. V. Jena, Y. Shamay, J. Shah, D. Roxbury, N. Paknejad, and D. A. Heller, “Photoluminescent carbon nanotubes interrogate the permeability of multicellular tumor spheroids,” Carbon N. Y., vol. 97, pp. 99–109, 2016.
 M. Benton, M. R. Hossan, P. R. Konari, and S. Gamagedara, “Effect of process parameters and material properties on laser micromachining of microchannels,” Micromachines, vol. 10, no. 2, p. 123, 2019.
 S. Mishra and V. Yadava, “Laser Beam MicroMachining (LBMM) - A review,” Opt. Lasers Eng., vol. 73, pp. 89–122, 2015.
 C. G. Khan Malek, “Laser Processing for Bio-microfluidics Applications (Part II),” Anal. Bioanal. Chem., vol. 385, no. 8, pp. 1362–1369, 2006.
 H. Klank, J. P. Kutter, and O. Geschke, “CO2-laser Micromachining and Back-end Processing for Rapid Production of PMMA-based Microfluidic Systems,” Lab Chip, vol. 2, no. 4, pp. 242–246, 2002.
 D. Yuan and S. Das, “Experimental and theoretical analysis of direct-write laser micromachining of polymethyl methacrylate by CO2 laser ablation,” J. Appl. Phys., vol. 101, no. 2, p. 024901, 2007.
 N. C. Nayak, Y. C. Lam, C. Y. Yue, and A. T. Sinha, “CO2-laser micromachining of PMMA: The effect of polymer molecular weight,” J. Micromechanics Microengineering, vol. 18, no. 9, 2008.
 X. Chen, J. Shen, and M. Zhou, “Rapid fabrication of a four-layer PMMA-based microfluidic chip using CO2-laser micromachining and thermal bonding,” J. Micromechanics Microengineering, vol. 26, no. 10, pp. 1–7, 2016.
 G. H. Lee, J. S. Lee, H. J. Oh, and S. H. Lee, “Reproducible construction of surface tension-mediated honeycomb concave microwell arrays for engineering of 3D microtissues with minimal cell loss,” PLoS One, vol. 11, no. 8, p. e0161026, 2016.
 J. M. Cha et al., “A novel cylindrical microwell featuring inverted-pyramidal opening for efficient cell spheroid formation without cell loss,” Biofabrication, vol. 9, no. 3, 2017.
 A. R. Thomsen et al., “A deep conical agarose microwell array for adhesion independent three-dimensional cell culture and dynamic volume measurement,” Lab Chip, pp. 179–189, 2018.
 S. W. Lee, S. Y. Jeong, T. H. Shin, J. Min, D. Lee, and G. S. Jeong, “A cell-loss-free concave microwell array based size-controlled multi-cellular tumoroid generation for anti-cancer drug screening,” PLoS One, 2019.
 Y. C. Chen et al., “Chemo-photothermal effects of doxorubicin/silica-carbon hollow spheres on liver cancer,” RSC Adv., vol. 8, no. 64, pp. 36775–36784, 2018.
 J. Bai, T.-Y. Tu, C. Kim, J. P. Thiery, and R. D. Kamm, “Identification of drugs as single agents or in combination to prevent carcinoma dissemination in a microfluidic 3D environment,” Oncotarget, vol. 6, no. 34, pp. 36603–36614, 2015.
 J. B. Lee et al., “A novel in vitro permeability assay using three-dimensional cell culture system,” J. Biotechnol., vol. 205, pp. 93–100, 2014.
 G. Fang, H. Lu, A. Law, D. Gallego-Ortega, D. Jin, and G. Lin, “Gradient-sized control of tumor spheroids on a single chip,” Lab Chip, vol. 19, no. 24, pp. 4093–4103, 2019.
 V. H. Huxley, F. E. Curry, and R. H. Adamson, “Quantitative fluorescence microscopy on single capillaries: α-lactalbumin transport,” Am. J. Physiol. - Hear. Circ. Physiol., vol. 252, no. 1, pp. H188-97, 1987.
 Y. T. Ho, G. Adriani, S. Beyer, P. T. Nhan, R. D. Kamm, and J. C. Y. Kah, “A Facile Method to Probe the Vascular Permeability of Nanoparticles in Nanomedicine Applications,” Sci. Rep., vol. 7, no. 1, pp. 1–13, 2017.
 J. Luo, X. Duan, Z. Chen, X. Ruan, Y. Yao, and T. Liu, “A laser-fabricated nanometer-thick carbon film and its strain-engineering for achieving ultrahigh piezoresistive sensitivity,” J. Mater. Chem. C, 2019.
 T.-Y. Tu, C.-Y. Chen, D.-S. Jong, and A. M. Wo, “An integrated electrophysiological and optical approach for ion channel study in a microfluidic system enabling intra- and extra-cellular solution exchange,” Sensors Actuators B Chem., vol. 185, pp. 496–503, Aug. 2013.
 C. Y. Chen, T. Y. Tu, D. S. Jong, and A. M. Wo, “Ion channel electrophysiology via integrated planar patch-clamp chip with on-demand drug exchange,” Biotechnol. Bioeng., 2011.
 C.-Y. Chen, T.-Y. Tu, C.-H. Chen, D.-S. Jong, and A. M. Wo, “Patch Clamping on Plane Glass-fabrication of Hourglass Aperture and High-yield Ion Channel Recording,” Lab Chip, vol. 9, no. 16, pp. 2370–2380, 2009.
 Š. Selimovi ć, F. Piraino, H. Bae, M. Rasponi, A. Redaelli, and A. Khademhosseini, “Microfabricated Polyester Conical Microwells for Cell Culture Applications,” Lab Chip, vol. 11, no. 14, pp. 2325–2332, 2011.
 Y. Y. Choi, B. G. Chung, D. H. Lee, A. Khademhosseini, J. H. Kim, and S. H. Lee, “Controlled-size embryoid body formation in concave microwell arrays,” Biomaterials, vol. 31, no. 15, pp. 4296–4303, 2010.
 J. Fukuda, S. Takahashi, T. Osaki, N. Mochizuki, and H. Suzuki, “Processing of nanolitre liquid plugs for microfluidic cell-based assays,” Sci. Technol. Adv. Mater., 2012.
 J. Bai, T. Y. Tu, C. Kim, J. P. Thiery, and R. D. Kamm, “Identification of drugs as single agents or in combination to prevent carcinoma dissemination in a microfluidic 3D environment,” Oncotarget, 2015.
 B. L. Khoo et al., “Liquid biopsy and therapeutic response: Circulating tumor cell cultures for evaluation of anticancer treatment,” Sci. Adv., 2016.
 S. Dasari and P. Bernard Tchounwou, “Cisplatin in cancer therapy: Molecular mechanisms of action,” European Journal of Pharmacology, vol. 740. pp. 364–378, 2014.
 S. A. Aldossary, “Review on pharmacology of cisplatin: Clinical use, toxicity and mechanism of resistance of cisplatin,” Biomed. Pharmacol. J., vol. 12, no. 1, pp. 7–15, 2019.
 X. M. Xu et al., “Combined anticancer activity of osthole and cisplatin in NCI-H460 lung cancer cells in vitro,” Exp. Ther. Med., vol. 5, no. 3, pp. 707–710, 2013.
 W. Yang et al., “Genomics of Drug Sensitivity in Cancer (GDSC): A resource for therapeutic biomarker discovery in cancer cells,” Nucleic Acids Res., vol. 41, no. D1, pp. D955-61, 2013.
 R. L. F. Amaral, M. Miranda, P. D. Marcato, and K. Swiech, “Comparative analysis of 3D bladder tumor spheroids obtained by forced floating and hanging drop methods for drug screening,” Front. Physiol., vol. 8, no. AUG, p. 605, 2017.
 J. H. Yeon, S. H. Chung, C. Baek, H. Hwang, and J. Min, “A Simple Pipetting-based Method for Encapsulating Live Cells into Multi-layered Hydrogel Droplets,” Biochip J., 2018.