此研究目的是建立人類肺氣管晶片以加速肺疾病模型的開發並找到適合的疾病治療模式。雖然目前絕大部分的體外人體肺細胞的研究都是建立於Transwell®組織培養皿，但是此細胞的培養程序繁複，細胞狀況或是結果都會因為人為手動操作而產生不同的結果，所以我們致力於開發半自動化培養系統，使研究人員減少人為操作上的失誤，並得到更穩定的數據。
我們將透過先進微加工技術結合雷射加工與逐層建構技術製作出多重材料結合之微環境晶片。此培養平台主要包括：細胞培養晶片、多功能儲存槽、以及流體控制系統。流體控制系統是由微型蠕動幫浦、夾管閥及微流道晶片所構成。不同的培養程序，像是細胞增生、細胞分化或是粘液的清洗等11項操作步驟，都可以透過電腦進行控制，以減少人為操作上所造成的誤差，並得到可再現性及穩定的肺組織模型。
我們首先透過HeLa cell與A549 來證明此系統的可行性，並進一步確認此系統可以在液-氣介面下長時間的培養人類細胞。我們也將動態流體環境應用於培養A549細胞中，預期此模擬生理環境的流體應力可以使上皮細胞分化和組織特異性功能，最後，透過人支氣管上皮細胞HBECs的免疫染色和組織切片顯示出在晶片中的緊密連接和假複層生長。除了支氣管組織培養之外，我們研發出的多種培養步驟的組織培養平台將亦適用於其他體外人類組織培養模型。

Here we describe the development of a human lung small airway on a chip to facilitate the lung disease model and suitable treatment. Although many standard procedures for the culture of a bronchial epithelial cell line are readily available using Transwell® inserts, the culture steps are considered to be labor intensive and the cell conditions are varied with the handling. In this work, we demonstrate a semi-automated platform for long-term mucociliary epithelium culture allowing the users to maintain the cell culture with minimum intervention. This highly integrated microfluidic-based platform was fabricated using a rapid prototyping technique based on laser patterned polymeric membranes/films/sheets and lamination to form the highly integrated bioreactor.
The platform mainly combines three parts: a cell culture device, a multi-functional reservoir, and a flow management system including a customized peristaltic pump, pinch valves, and fluid network. The culture operation mode allowing proliferation, differentiation and mucus washing, can be easily switched by this computer-controlled system. With this programmed cultivation, it is possible to obtain reproducible and consistent lung model and reduce the deviations from the manual operation.
We first demonstrated the feasibility of our system by culturing HeLa cell and A549 cells to check the long-term cultivated ability under air-liquid interface without contamination. The dynamic flow was applied to the A549 cell culture in both submerged and air-lifted conditions. Then, the organotypic analysis including the immunostaining and tissue sectioning results of the human bronchial epithelial cells (HBECs) shown the tight junction and a pseudostratified growth within our device. In addition to bronchial tissue culture, the versatile combination of culture steps makes the culture platform suitable to other in vitro human tissue culture models.

Chapter 1: Introduction and Literature Review------------1
1-1 Lung characteristic------------------------------1
1-1-1 Background---------------------------------------1
1-1-2 Lung structure-----------------------------------2
1-1-3 Particle deposition------------------------------3
1-1-4 Defense mechanism--------------------------------3
1-1-5 Limitation of animal models----------------------4
1-2 Microchip----------------------------------------5
1-2-1 Microfluidic-------------------------------------5
1-2-2 Organ chip---------------------------------------6
1-2-3 Driving force in the microsystem-----------------6
1-3 Fabrication and material of microfluidic device--7
1-3-1 Material-----------------------------------------7
1-3-2 Manufacturing------------------------------------7
1-4 Cell culture-------------------------------------8
1-4-1 Transwell®---------------------------------------8
1-4-2 Human bronchial epithelium cell culture----------8
1-4-3 Cell culture challenge---------------------------9
1-4-4 Characteristic of epithelial tissue--------------10
1-4-5 Current study------------------------------------11
1-5 Motivation and purpose---------------------------11
Chapter 2: Materials and methods-------------------------13
2-1 Experimental flow chart--------------------------13
2-2 Platform design and fabrication------------------14
2-2-1 Peristaltic pump---------------------------------14
2-2-2 Fabrication--------------------------------------15
2-2-3 Fluid control system-----------------------------17
2-2-4 Culture device-----------------------------------19
2-2-5 Liquid Flow Meter SLI----------------------------21
2-3 Cell culture-------------------------------------22
2-3-1 Microscope---------------------------------------22
2-3-2 Preprocedure before the culture------------------22
2-3-3 Cell culture-------------------------------------22
2-3-4 Live/Dead cell viability assay-------------------25
2-3-5 Tissue sectioning and immunostaining-------------25
Chapter 3. Result----------------------------------------27
3-1 Fabrication of the multifunctional platform------27
3-1-1 Flow stability test------------------------------27
3-1-2 Bronchiolar flow chart – ink flow test-----------28
3-2 Sterilization process----------------------------30
3-3 Initial testing for cell proliferation assay-----31
3-4 A549 cell in submerged, air-lift, and dynamic flow conditions-----------------------------------------------32
3-5 Human bronchial epithelial cell culture----------35
3-6 An obstacle for the dynamical culture------------43
3-7 3-D structures-----------------------------------45
Chapter 4. Conclusion and future works-------------------48
Chapter 5. References------------------------------------50

1. M.polak, A.E.B.a.J., Lungs, in Principles of Tissue Engineering, 3rd Edition, L. Lanza, and Vacanti, Editor. 2007.
2. Loira-Pastoriza, C., J. Todoroff, and R. Vanbever, Delivery strategies for sustained drug release in the lungs. Advanced drug delivery reviews, 2014. 75: p. 81-91.
3. Bajaj, P., et al., Advances and challenges in recapitulating human pulmonary systems: at the cusp of biology and materials. ACS Biomaterials Science & Engineering, 2016. 2(4): p. 473-488.
4. Weibel, E.R., B. Sapoval, and M. Filoche, Design of peripheral airways for efficient gas exchange. Respiratory physiology & neurobiology, 2005. 148(1-2): p. 3-21.
5. Horsfield, K. and G. Cumming, Morphology of the bronchial tree in man. Journal of applied physiology, 1968. 24(3): p. 373-383.
6. Hussain, M., P. Madl, and A. Khan, Lung deposition predictions of airborne particles and the emergence of contemporary diseases, Part-I. Health, 2011. 2(2): p. 51-59.
7. Seaton, A., et al., Particulate air pollution and acute health effects. The lancet, 1995. 345(8943): p. 176-178.
8. Ruge, C.A., J. Kirch, and C.-M. Lehr, Pulmonary drug delivery: from generating aerosols to overcoming biological barriers—therapeutic possibilities and technological challenges. The Lancet Respiratory Medicine, 2013. 1(5): p. 402-413.
9. Wanner, A., The role of mucus in chronic obstructive pulmonary disease. Chest, 1990. 97(2): p. 11s-15s.
10. Geiser, M., Update on macrophage clearance of inhaled micro-and nanoparticles. Journal of aerosol medicine and pulmonary drug delivery, 2010. 23(4): p. 207-217.
11. Lam, J.K.-W., W. Liang, and H.-K. Chan, Pulmonary delivery of therapeutic siRNA. Advanced drug delivery reviews, 2012. 64(1): p. 1-15.
12. Monteiro, A. and R.L. Smith, Bronchial tree Architecture in Mammals of Diverse Body Mass. International Journal of Morphology, 2014. 32(1).
13. Beebe, D.J., G.A. Mensing, and G.M. Walker, Physics and applications of microfluidics in biology. Annual review of biomedical engineering, 2002. 4(1): p. 261-286.
14. Pamula, V.K. and K. Chakrabarty. Cooling of integrated circuits using droplet-based microfluidics. in Proceedings of the 13th ACM Great Lakes symposium on VLSI. 2003. ACM.
15. Yeo, L.Y. and J.R. Friend, Ultrafast microfluidics using surface acoustic waves. Biomicrofluidics, 2009. 3(1): p. 012002.
16. Esch, E.W., A. Bahinski, and D. Huh, Organs-on-chips at the frontiers of drug discovery. Nature reviews Drug discovery, 2015. 14(4): p. 248.
17. Frangos, J., L. McIntire, and S. Eskin, Shear stress induced stimulation of mammalian cell metabolism. Biotechnology and bioengineering, 1988. 32(8): p. 1053-1060.
18. Nollert, M., S. Diamond, and L. McIntire, Hydrodynamic shear stress and mass transport modulation of endothelial cell metabolism. Biotechnology and Bioengineering, 1991. 38(6): p. 588-602.
19. Halldorsson, S., et al., Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices. Biosensors and Bioelectronics, 2015. 63: p. 218-231.
20. Bhise, N.S., et al., Organ-on-a-chip platforms for studying drug delivery systems. Journal of Controlled Release, 2014. 190: p. 82-93.
21. Maschmeyer, I., et al., A four-organ-chip for interconnected long-term co-culture of human intestine, liver, skin and kidney equivalents. Lab on a Chip, 2015. 15(12): p. 2688-2699.
22. Benam, K.H., et al., Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro. Nature methods, 2016. 13(2): p. 151.
23. Trieu, D., T.K. Waddell, and A.P. McGuigan, A microfluidic device to apply shear stresses to polarizing ciliated airway epithelium using air flow. Biomicrofluidics, 2014. 8(6): p. 064104.
24. Bousse, L., et al., Electrokinetically controlled microfluidic analysis systems. Annual review of biophysics and biomolecular structure, 2000. 29(1): p. 155-181.
25. Skafte-Pedersen, P., et al., Multi-channel peristaltic pump for microfluidic applications featuring monolithic PDMS inlay. Lab on a Chip, 2009. 9(20): p. 3003-3006.
26. Grass, B., et al., A new PMMA-microchip device for isotachophoresis with integrated conductivity detector. Sensors and Actuators B: Chemical, 2001. 72(3): p. 249-258.
27. Shin, Y.S., et al., PDMS-based micro PCR chip with parylene coating. Journal of Micromechanics and Microengineering, 2003. 13(5): p. 768.
28. Unger, M.A., et al., Monolithic microfabricated valves and pumps by multilayer soft lithography. Science, 2000. 288(5463): p. 113-116.
29. Whitesides, G.M. and A.D. Stroock, Flexible methods for microfluidics. Phys. Today, 2001. 54(6): p. 42-48.
30. Klank, H., J.P. Kutter, and O. Geschke, CO 2-laser micromachining and back-end processing for rapid production of PMMA-based microfluidic systems. Lab on a Chip, 2002. 2(4): p. 242-246.
31. Velden, V. and H. Versnel, Bronchial epithelium: morphology, function and pathophysiology in asthma. European cytokine network, 1998. 9(4): p. 585-597.
32. Kikuchi, T., et al., Differentiation-dependent responsiveness of bronchial epithelial cells to IL-4/13 stimulation. American Journal of Physiology-Lung Cellular and Molecular Physiology, 2004. 287(1): p. L119-L126.
33. Grainger, C.I., et al., Culture of Calu-3 cells at the air interface provides a representative model of the airway epithelial barrier. Pharmaceutical research, 2006. 23(7): p. 1482-1490.
34. Fanning, A.S., et al., The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. Journal of Biological Chemistry, 1998. 273(45): p. 29745-29753.
35. Schneeberger, E.E. and R.D. Lynch, The tight junction: a multifunctional complex. American Journal of Physiology-Cell Physiology, 2004. 286(6): p. C1213-C1228.
36. Ostrowski, L.E., et al., A Proteomic Analysis of Human Cilia Identification of Novel Components. Molecular & Cellular Proteomics, 2002. 1(6): p. 451-465.
37. Blank, F., et al., An optimized in vitro model of the respiratory tract wall to study particle cell interactions. Journal of aerosol medicine, 2006. 19(3): p. 392-405.