此研究目的是建立人類肺氣管晶片以加速肺疾病模型之開發，進而研究出適合的疾病治 療方式 。目前大多數的體外人體肺細胞之研究均是於 Transwell® 組織培養皿上，然而因為 人體肺細胞的繁複培養程序，且細胞狀況亦會因為人為手動操作而產生不同的結果，因此我 們致力於開發自動化培養系統，使研究人員減少人為操作上的失誤，進而得到更穩定之研究 成果。
透過先進微加工技術結合雷射加工與逐層建構技術，製作多重材料結合之微流體流體器 官培養裝置。此培養平台主要包括:細胞培養晶片、2D 與 3D 之微流道系統、以及 Arduino 微流體控制系統。透過微流體控制系統，得以創造對應不同培養程序之所需環境，如細胞增 生、細胞分化或是粘液的清洗等等，進而實現自動化培養細胞之理念，並得到具再現性及穩 定性之肺組織模型。
透過 A549 與 HBEC 來證明此系統的可行性，並進一步確認此系統可以在液-氣介面下長 時間地培養人體細胞是此研究之目標。未來也會將動態流體環境應用於細胞培養中，預期此 模擬生理環境之流體可以更完整地建構人體體內環境，使後續研究能更符合人體真實反應。 除了支氣管組織培養之外，利用微流體創造特定所需生長環境後，我們研發出的組織培養平 台將亦適用於其他體外人類組織培養模型。

Microfluidic components have been applied to lab-on-a-chip (LOC) for rapid and more
efficient performance for years. However, organ-level cell culture still has limitations of time- consuming, labor-intensive and risk of contamination. For instance, lung cells take at least one month to proliferate and differentiate into tissue.
In this work, we introduced a Plug-and-Play design in the lung tissue culture platform to eliminate the risk of contamination and to improve the cell culture efficiency. Besides, we make the culture platform operate automatically in specific cell culture processes, such as mucus washing and air-lifting. The Plug-and-Play tissue culture platform included a cell culture chip, a fluid control base, peristaltic pumps and pinch-type valves controlled by a computer. This cell culture chip has two independent chambers separated by a microporous membrane for lung cell proliferation and differentiation, requiring a cell culture environment of liquid-liquid interphase and air-liquid interphase, respectively. The Plug-and-Play culture platform mounted with miniaturized devices can simplify the tissue culture procedure, which only requires inserting the lung tissue culture chip at the beginning and removing it after finishing.
As a result, we demonstrated the cell culture platform is bio-compatible by stationary A549 cell culture. Besides, human bronchial epithelial cell (HBEC) was cultured in the culture chip to test the function of differentiation. The static culture for HBEC performs the same differentiation result with the culture in a Transwell® culture plate. With this culture platform, we can culture different cells by designing the required procedures. Furthermore, this platform may be applied to other fields, such as tissue engineering and regenerative medicine.

Chapter 1: Introduction and Literature Review .................. 1
1-1 Development of tissue culture ............................................................................................... 1 1-1-1 Limitation of animal model.................................................................................. 1
1-1-2 Current tissue culture technology ......................................................................... 1
1-1-3 2D and 3D Microfluidic culture ........................................................................... 2
1-2 2D Chip cell culture .............................................................................................................. 3 1-2-1 Fabrication method .............................................................................................. 3
1-2-2 Material ............................................................................................................... 4
1-2-3 Automatic tissue culture ....................................................................................... 5
1-2-4 Human bronchial epithelial cells (HBECs)........................................................... 5
1-2-5 Lung-on-chip review............................................................................................ 6
1-3 Motivation and purpose......................................................................................................... 7
1-3-1 Challenge for the cell culture platform ................................................................. 7 1-3-2 Automatic Plug-and Play platform for long-term tissue culture ............................ 7
Chapter 2: Design, Material, and Method........................... 9
2-1 Microfluidic system .............................................................................................................. 9 2-1-1 Material ............................................................................................................... 9
2-1-2 Fabrication principle .......................................................................................... 10
2-1-3 Microfluidic system overview ............................................................................ 10
2-1-4 Cell culture chip and Chip container .................................................................. 12 2-1-5 Reservoir and Fluid control base ........................................................................ 14 2-1-6 Peristaltic pump ................................................................................................. 15
2-2 Arduino control system ....................................................................................................... 16
2-2-1 Circuit design..................................................................................................... 16 2-2-2 Solenoid valves.................................................................................................. 17 2-2-3 Installation of Arduino system on microfluidic system ....................................... 18
2-3 Integration platform and cell culture.................................................................................... 18 2-3-1 Operation mechanism ........................................................................................ 18
2-3-2 Before cell culture – Sterilization....................................................................... 19
2-3-3 Cell culture (A549, HBEC)................................................................................ 20
2-4 Comparing method between cell culture chip and Transwell® ............................................. 21 2-4-1 A549 with Green Fluorescent Protein (GFP) ...................................................... 21
2-4-2 HBEC ................................................................................................................ 21
Chapter 3: Result and Discussion ..................................... 23
3-1 Verification of the cell culture platform ............................................................................... 23 3-1-1 Platform design feature ...................................................................................... 23
3-1-2 Flow stability test............................................................................................... 24
3-2 Solenoid valves performance test ........................................................................................ 25 3-2-1 Fluid control test................................................................................................ 25
3-2-2 Long-time operation test .................................................................................... 27
3-3 Bio-compatibility test.......................................................................................................... 28 3-3-1 A549 static culture ............................................................................................. 28
3-3-2 HBEC static culture ........................................................................................... 30
3-3-3 Differentiation features with and without growth kits......................................... 35
3-4 Dynamic culture (A549) ..................................................................................................... 36 3-4-1 A549 GFP distribution ....................................................................................... 36
Chapter 4: Conclusion and Future works.......................... 38
4-1 Conclusion.......................................................................................................................... 38
4-2 Dynamic cell culture for HBECs......................................................................................... 38 4-3 Medium freshness measurement.......................................................................................... 38 4-4 High throughput cell culture................................................................................................ 39
Chapter 5: References....................................................... 40

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