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系統識別號 U0026-0907202014391500
論文名稱(中文) 建構具高度重複之微孔陣列平台於高通量腫瘤球體培養和藥物評估之研究
論文名稱(英文) Development of a highly reproducible microwell array platform for high-throughput tumor spheroid culture and drug assessment
校院名稱 成功大學
系所名稱(中) 生物醫學工程學系
系所名稱(英) Department of BioMedical Engineering
學年度 108
學期 2
出版年 109
研究生(中文) 巫光偉
研究生(英文) Kuang-Wei Wu
學號 P86074064
學位類別 碩士
語文別 英文
論文頁數 52頁
口試委員 指導教授-涂庭源
口試委員-王應然
口試委員-葉思沂
中文關鍵字 三維腫瘤培養  多細胞腫瘤球體  體外腫瘤模型  微孔  通透係數  高通量藥物篩選  順鉑 
英文關鍵字 Three-dimensional (3D) tumor culture  multicellular tumor spheroids (MCTS)  in vitro tumor model  microwell  permeability coefficient  high-throughput drug screening  cisplatin 
學科別分類
中文摘要 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。
英文摘要 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.
論文目次 摘要 I
Abstract II
誌謝 IV
Contents VI
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
References 46

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