體外細胞培養模型是醫學生物技術工程的基礎研究工具，其被應用於藥物篩選等癌症相
關研究。目前被廣泛使用的模型，如:細胞培養角瓶或培養皿等，都因靜態培養條件不能準
確地模擬體內腫瘤環境，而導致篩選結果不盡準確。
在此研究中，透過雷射切割及逐層堆疊技術而建構出一微灌輸培養平台。這個平台包
含了三個獨立運作的液壓驅動系統，以同時進行三組細胞培養與藥物篩選。此外，這樣的系
統組合可以實現可控制的剪切力與應變力;而各個系統都包含三個部分:一個負責維持運作
的液壓驅動微幫浦、一對防止逆流的可更換微單向閥，以及一可自由拆卸的培養器。並且三
個系統都由同一個蠕動式幫浦提供總驅動力。此平台先透過單純的細胞培養實驗以確保細胞
在動態條件下依然能正常增殖;最後，在藥物測試的實驗中比較了靜態與動態不同條件下，
細胞的型態變化以及生存率等表現。
未來，將整合更多系統單位以建立更完善的平台，並改良成三維細胞培養模型，更準
確地進行細胞培養與藥物測試，以確立此平台的開發潛力。

Cell culture has been regarded as a foundational tool for medical biotechnology, for instance, it is widely applied in cancer research for drug screening. Conventional in vitro culture models, such as flasks or petri dishes, have been mostly used so far; yet static conditions might make it hard to simulate dynamic environment of the human system, resulting in an inaccurate screening outcome.
Here, a microfluidic platform operating in dynamic conditions was fabricated by laser cutting and additive lamination manufacturing. In this platform, three hydraulically-driven perfusion systems were included, suggesting three models for culturing and screening could be done simultaneously. In addition, controllable shear stress and mechanical strain were achievable due to the combination of perfusion systems, where each system consisted of three parts: a hydraulically- driven pump to keep the whole system working automatically, a pair of micro-valves for ensuring medium flowed one direction only, and a bioreactor where the cells were cultured and treated. The feasibility of platform was first examined by a culture experiment to ensure the normal proliferation. Finally, cell morphology and viability under static and different dynamic conditions were compared. In the future, more system units will be integrated to establish a more complete platform and improved into a three-dimensional model for more accurate cell culture and drug screening to ensure the potential of this platform.

Abstract _____________________________I
摘要 ________________________________II
序言 ________________________________III
Contents ____________________________IV
List of Illustrations ____________________VII
List of Tables ________________________X
Chapter 1. Introduction ________________1
1-1 Background.............................................1
1-1-1 Cancer statistics..................................1
1-1-2 Cancer therapies..................................2
1-2 Conventional Screening Models..............4
1-2-1 In vitro models .....................................4
1-2-2 In vivo models......................................5
1-3 Dynamic Culture......................................6
1-4 Microfluidics Technology ........................8
1-4-1 Lab-on-chip system..............................8
1-4-2 Micro-perfusion system ......................9
1-5 Purposes ..................................................11
Chapter 2. Materials and Methods _________12
2-1 Materials ..................................................12
2-1-1 Polyethylene Terephthalate (PET) ..........12
2-1-2 Poly(methyl methacrylate) (PMMA) .........13
2-1-3 Polydimethylsiloxane (PDMS) Membrane...13
2-1-4 O’ring .......................................................14
2-1-5 Silicon Tubing ...........................................15
2-2 Platform Fabrication and Device Design........15
2-2-1 Fabrication Process ...................................16
2-2-2 Peristaltic Pump ..........................................18
2-2-3 Concept and Principle .................................19
2-2-4 Device Versions and Overview ....................21
2-2-5 Micro-pump .................................................26
2-2-6 Micro-valve...................................................30
2-2-7 Bioreactor.......................................................33
2-2-8 Platform Setup and Operating Procedure......34
2-3 Flow Measurements...........................................36
2-3-1 Flow pattern Analysis.......................................37
2-3-2 Flow Rate and Stability Test .............................37
2-3-3 Flow Rate Test of a Peristaltic Pump ................38
2-4 A549 Cell Culture .................................................38
2-4-1 Platform Sterilization .........................................39
2-4-2 Static Culture.....................................................40
2-4-3 Dynamic Culture ...............................................40
2-5 Drug Screening .....................................................41
2-5-1 Screening Procedure .........................................41
2-5-2 Live/Dead Assay ................................................42
2-5-3 Counting Cell Kit-8 (CCK-8) Cytotoxicity Assay ...42
2-5-4 Diffusion Test.........................................................43
Chapter 3. Results and Discussion __________________44
3-1 Flow Measurements...................................................44
3-1-1 Flow Pattern ...........................................................44
3-1-2 Stability and Flow Rate Test ...................................46
3-1-3 Comparison: Peristaltic Pump versus Platform ......48
3-2 Cell Culture.................................................................49
3-2-1 Static Culture...........................................................49
3-2-2 Dynamic Culture .....................................................50
3-3 Drug Screening ...........................................................52
3-3-1 Qualitative Analysis ..................................................52
3-3-2 Quantitative Analysis ...............................................54
3-4 Discussion....................................................................56
3-4-1 Diffusion Curve.........................................................56
3-4-2 Comparison: Serum versus Serum-free ..................57
3-4-3 Impact of Flow Stress ..............................................59
Chapter 4. Conclusions and Future Work ______________63
4-1 Conclusions..................................................................63
4-2 Future Work .................................................................64
4-2-1 Screening Assays of More Cell Lines .......................64
4-2-2 Expansion of Device Units .......................................64
4-2-3 3D Screening............................................................65
4-2-4 Drug Screening with Serum......................................65
Chapter 5 References ______________________________66

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