臺灣博碩士論文加值系統

致謝 i
目錄 iii
圖目錄 vii
表目錄 ix
中文摘要 x
英文摘要 xi
縮寫對照表 xiii
第一章、緒論（Introduction） 14
1.1 傳統藥物吸收模型的限制 (Limitations of Traditional Drug Absorption Models） 14
1.1.1體外模型的限制（Limitations of In Vitro Models） 14
1.1.2離體模型的限制（Limitations of Ex Vivo Models） 14
1.1.3活體模型的限制（Limitations of In Vivo Models） 15
1.2 器官晶片技術（Organ-on-a-Chip technology） 16
1.3 腸道晶片系統的設計原則（Design Principles of Gut-on-a-Chip Systems） 19
1.3.1 生物學需求（Biological Considerations） 19
1.3.2晶片材料與微流控設計（Chip Material and Microfluidics Design） 19
1.3.3組織工程與細胞培養（Tissue Engineering and Cell Culture） 20
1.4 Transwell 系統的基本原理 （Basic Principles of the Transwell System） 21
1.4.1 Transwell 系統在腸道藥物吸收研究中的應用(Application of Transwell System in intestinal drug absorption research) 21
1.4.2 Transwell 系統的實驗方法與測試標準（Experimental Methodology and Evaluation Standards of the Transwell System） 22
1.5 Transwell 系統與腸道晶片的比較( Comparison between Transwell and Gut-on-a-chip System) 24
第二章、研究目的（Aims of study） 25
第三章、實驗材料及方法（Materials and methods） 27
3.1材料與儀器（Materials and instruments） 27
3.1.1 材料（Materials） 27
3.1.2 實驗儀器（Instruments） 29
3.2 細胞培養（Cell culture） 30
3.3 ELVEFLOW系統設置（ELVEFLOW system setting） 30
3.4 腸道晶片的方法建立（Establishment of Gut-on-a-Chip Protocols） 31
3.4.1 藥物滲透性試驗（Drug Permeability Test） 33
3.5 Transwell 系統建立（Transwell System setup） 34
3.5.1 Transwell 藥物滲透度測量（Evaluation of Drug Permeability Using the Transwell System） 34
3.6 BCS Class藥物的分析方法與確效（Analytical Methods and Validation for BCS-Class Drugs） 35
3.6.1 Caffeine的HPLC分析方法（HPLC Analytical Method for Caffeine） 35
3.6.2 Minoxidil的HPLC分析方法（HPLC Analytical Method for Minoxidil） 36
3.6.3 Acetaminophen的HPLC分析方法（HPLC Analytical Method for Acetaminophen） 37
3.6.4 Abiraterone Acetate的分析方法（HPLC Analytical Method for Abiraterone Acetate） 39
3.7形態學（Morphology） 42
3.7.1 Caco-2 細胞免疫螢光染色（Immunofluorescence Staining of Caco-2 Cells） 42
第四章、結果（Result） 43
4.1 Transwell 系統中上皮屏障完整性的 TEER 測定 (TEER Measurement of Epithelial Barrier Integrity in Transwell System) 43
4.2分析方法的建立與確校 44
4.2.1 Caffeine 之 HPLC 分析方法確效（Analytical Method for Caffeine） 44
4.2.2 Minoxidil 之 HPLC 分析方法確效（Analytical Method for Minoxidil） 47
4.2.3 Acetaminophen 之 HPLC 分析方法確效（Analytical Method for Acetaminophen） 50
4.3 Tomocube 53
4.4 晶片系統與Transwell系統的藥物通透性比較(Permeability Comparison between Chip system & Transwell system) 59
4.4.1 晶片與Transwell Caffeine之 Papp比較（Comparison of Caffeine Papp Between Chip and Transwell Systems） 59
4.4.2 晶片與Transwell Minxoidil之 Papp比較（Comparison of Minxoidil Papp Between Chip and Transwell Systems） 61
4.4.3 晶片與Transwell Acetaminophen之 Papp比較（Comparison of Acetaminophen Papp Between Chip and Transwell Systems） 63
4.5 形態學(Morphology） 65
第五章、討論 (Discussion) 71
第六章、結論 (Conclusion) 73
第七章、參考文獻(Reference) 74

圖目錄
Figure 1 Comparison of In Vitro, Ex Vivo, and In Vivo Models[2] 13
Figure 2 Organ-on-chip[30] 16
Figure 3 Gut-on-a-Chip Schematic[31] 18
Figure 4 Flow chart 24
Figure 5 Timeline of Gut-on-Chip System Preparation 30
Figure 6 Schematic of TEER Measurement Setup 32
Figure 7 TEER Assessment of Epithelial Barrier Integrity in Various Groups 41
Figure 8 HPLC chromatograms showed the retention time peaks of Caffeine 42
Figure 9 Intraday calibration curve of drug solution. 43
Figure 10 Interday calibration curve of drug solution. 43
Figure 11 HPLC chromatograms showed the retention time peaks of Minoxidil 45
Figure 12 Intraday calibration curve of drug solution. 46
Figure 13. Interday calibration curve of drug solution. 46
Figure 14 HPLC chromatograms showed the retention time peaks of Acetaminophen 48
Figure 15 Intraday calibration curve of drug solution. 49
Figure 16 Interday calibration curve of drug solution. 49
Figure 17 Tomocube 3D image of Caco-2 cells cultured under static conditions in a microfluidic chip 52
Figure 18 Z-axis cross-sectional image of Caco-2 cells cultured under static conditions 52
Figure 19 Tomocube 3D image of Caco-2 cells cultured under dynamic flow conditions. 54
Figure 20 Z-axis cross-sectional view of Caco-2 cells cultured under dynamic flow conditions 54
Figure 21 Tomocube Image Showing Moderate Intracellular Changes 56
Figure 22 Caffeine Permeability in Gut-on-Chip With and Without Cellular Barrier 58
Figure 23 Comparison of Caffeine Permeability between Transwell and 58
Figure 24 Minoxidil Permeability in Gut-on-Chip With and Without Cellular Barrier 60
Figure 25 Comparison of Minoxidil Permeability between Transwell and 60
Figure 26 Acetaminophen Permeability in Gut-on-Chip With and Without Cellular Barrier 62
Figure 27 Comparison of Acetaminophen Permeability between Transwell and Gut-on-Chip Models 62
Figure 28 Caco-2 confocal Z-stack Brightfield/Merged image 64
Figure 29 Caco-2 confocal Z-stack FITC(E-cadherin)/PI Image 65
Figure 30 HUVEC Cell Monolayers with FITC and DAPI 67
Figure 31 Comparison of Apparent Permeability Coefficient (Papp) of Caffeine Across Different Models 68
表目錄
Table 1. Parameters of Caffeine HPLC analytical method. 34
Table 2 Parameters of Minoxidil HPLC analytical method 35
Table 3 Parameters of Acetaminophen HPLC analytical method 36
Table 4 Parameters of mass spectrometry 37
Table 5 Conditions of UPLC-MS/MS. 38
Table 6 Gradient condition of UPLC-MS/MS. 38
Table 7 Optimized MRM parameters of Abiraterone acetate and internal standard. 39
Table 8 Intraday accuracy and precision (n=3) of Caffeine solution. 44
Table 9 Interday accuracy and precision (n=3) of Caffeine solution. 44
Table 10 Intraday accuracy and precision (n=3) of Minoxidil solution 47
Table 11 Interday accuracy and precision (n=3) of Minoxidil solution 47
Table 12 Intraday accuracy and precision (n=3) of Acetaminophen solution 50
Table 13 Interday accuracy and precision (n=3) of Acetaminophen solution 50

1.Dahlgren, D. and H. Lennernas, Intestinal Permeability and Drug Absorption: Predictive Experimental, Computational and In Vivo Approaches. Pharmaceutics, 2019. 11(8).
2.Xu, Y., et al., An overview of in vitro, ex vivo and in vivo models for studying the transport of drugs across intestinal barriers. Adv Drug Deliv Rev, 2021. 175: p. 113795.
3.Gamboa, J.M. and K.W. Leong, In vitro and in vivo models for the study of oral delivery of nanoparticles. Adv Drug Deliv Rev, 2013. 65(6): p. 800-10.
4.Rozehnal, V., et al., Human small intestinal and colonic tissue mounted in the Ussing chamber as a tool for characterizing the intestinal absorption of drugs. Eur J Pharm Sci, 2012. 46(5): p. 367-73.
5.Luo, Z., et al., Ex vivo and in situ approaches used to study intestinal absorption. J Pharmacol Toxicol Methods, 2013. 68(2): p. 208-216.
6.Costa, C.M., et al., A Critical Review on In Vitro and Ex Vivo Models of the Intestinal Epithelium of Humans and Monogastric Animals. Gastrointestinal Disorders, 2024. 6(1): p. 337-358.
7.Alam, M.A., F.I. Al-Jenoobi, and A.M. Al-Mohizea, Everted gut sac model as a tool in pharmaceutical research: limitations and applications. J Pharm Pharmacol, 2012. 64(3): p. 326-36.
8.Sjogren, E., et al., In vivo methods for drug absorption - comparative physiologies, model selection, correlations with in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur J Pharm Sci, 2014. 57: p. 99-151.
9.Srivastava, S.K., et al., Organ-on-chip technology: Opportunities and challenges. Biotechnol Notes, 2024. 5: p. 8-12.
10.Farhang Doost, N. and S.K. Srivastava, A Comprehensive Review of Organ-on-a-Chip Technology and Its Applications. Biosensors (Basel), 2024. 14(5).
11.Leung, C.M., et al., A guide to the organ-on-a-chip. Nature Reviews Methods Primers, 2022. 2(1).
12.Zhao, Q., et al., Mechanical Strain-Enabled Reconstitution of Dynamic Environment in Organ-on-a-Chip Platforms: A Review. Micromachines (Basel), 2021. 12(7).
13.Xu, F., et al., Architecture design and advanced manufacturing of heart-on-a-chip: scaffolds, stimulation and sensors. Microsyst Nanoeng, 2024. 10: p. 96.
14.Marsano, A., et al., Beating heart on a chip: a novel microfluidic platform to generate functional 3D cardiac microtissues. Lab Chip, 2016. 16(3): p. 599-610.
15.Jayne, R.K., et al., Direct laser writing for cardiac tissue engineering: a microfluidic heart on a chip with integrated transducers. Lab Chip, 2021. 21(9): p. 1724-1737.
16.Kim, H.J., et al., Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow. Lab Chip, 2012. 12(12): p. 2165-74.
17.Garcia-Gutierrez, E. and P.D. Cotter, Relevance of organ(s)-on-a-chip systems to the investigation of food-gut microbiota-host interactions. Crit Rev Microbiol, 2022. 48(4): p. 463-488.
18.Humayun, M., C.W. Chow, and E.W.K. Young, Microfluidic lung airway-on-a-chip with arrayable suspended gels for studying epithelial and smooth muscle cell interactions. Lab Chip, 2018. 18(9): p. 1298-1309.
19.Benam, K.H., et al., Matched-Comparative Modeling of Normal and Diseased Human Airway Responses Using a Microengineered Breathing Lung Chip. Cell Syst, 2016. 3(5): p. 456-466 e4.
20.Benam, K.H., et al., Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro. Nat Methods, 2016. 13(2): p. 151-7.
21.Deng, J., et al., Engineered Liver-on-a-Chip Platform to Mimic Liver Functions and Its Biomedical Applications: A Review. Micromachines (Basel), 2019. 10(10).
22.Deng, J., et al., A liver-chip-based alcoholic liver disease model featuring multi-non-parenchymal cells. Biomed Microdevices, 2019. 21(3): p. 57.
23.Vernetti, L.A., et al., A human liver microphysiology platform for investigating physiology, drug safety, and disease models. Exp Biol Med (Maywood), 2016. 241(1): p. 101-14.
24.Musah, S., et al., Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip. Nat Biomed Eng, 2017. 1.
25.Jang, K.J., et al., Human kidney proximal tubule-on-a-chip for drug transport and nephrotoxicity assessment. Integr Biol (Camb), 2013. 5(9): p. 1119-29.
26.Huang, W., et al., Revolutionizing nephrology research: expanding horizons with kidney-on-a-chip and beyond. Front Bioeng Biotechnol, 2024. 12: p. 1373386.
27.Carvalho, V., et al., Advancing Blood–Brain Barrier-on-a-Chip Models Through Numerical Simulations. BioChip Journal, 2024. 18(4): p. 546-565.
28.Reshma, S., et al., Blood brain barrier-on-a-chip to model neurological diseases. Journal of Drug Delivery Science and Technology, 2023. 80.
29.Augustine, R., et al., Bioengineered microfluidic blood-brain barrier models in oncology research. Transl Oncol, 2021. 14(7): p. 101087.
30.Driver, R. and S. Mishra, Organ-On-A-Chip Technology: An In-depth Review of Recent Advancements and Future of Whole Body-on-chip. BioChip Journal, 2022. 17(1): p. 1-23.
31.Donkers, J.M., J.I.v.d. Vaart, and E. Van de Steeg, 2023.
32.Ahadian, S., et al., Organ-On-A-Chip Platforms: A Convergence of Advanced Materials, Cells, and Microscale Technologies. Adv Healthc Mater, 2018. 7(2).
33.Kasendra, M., et al., Development of a primary human Small Intestine-on-a-Chip using biopsy-derived organoids. Sci Rep, 2018. 8(1): p. 2871.
34.Maurer, M., et al., A three-dimensional immunocompetent intestine-on-chip model as in vitro platform for functional and microbial interaction studies. Biomaterials, 2019. 220: p. 119396.
35.Auner, A.W., et al., Chemical-PDMS binding kinetics and implications for bioavailability in microfluidic devices. Lab Chip, 2019. 19(5): p. 864-874.
36.Tsao, C.W., Polymer Microfluidics: Simple, Low-Cost Fabrication Process Bridging Academic Lab Research to Commercialized Production. Micromachines (Basel), 2016. 7(12).
37.Vera, D., et al., A 3D bioprinted hydrogel gut-on-chip with integrated electrodes for transepithelial electrical resistance (TEER) measurements. Biofabrication, 2024. 16(3).
38.Su, W., et al., Microfluidics-assisted electrospinning of aligned nanofibers for modeling intestine barriers. PeerJ, 2022. 10: p. e13513.
39.Karamanos, N.K., et al., A guide to the composition and functions of the extracellular matrix. FEBS J, 2021. 288(24): p. 6850-6912.
40.Naomi, R., et al., Natural-Based Biomaterial for Skin Wound Healing (Gelatin vs. Collagen): Expert Review. Polymers (Basel), 2021. 13(14).
41.Hubatsch, I., E.G. Ragnarsson, and P. Artursson, Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat Protoc, 2007. 2(9): p. 2111-9.
42.Pires, C.L., et al., Re-Use of Caco-2 Monolayers in Permeability Assays-Validation Regarding Cell Monolayer Integrity. Pharmaceutics, 2021. 13(10).
43.Hilgers, A.R., R.A. Conradi, and P.S. Burton, Caco-2 cell monolayers as a model for drug transport across the intestinal mucosa. Pharm Res, 1990. 7(9): p. 902-10.
44.Volpe, D.A., Drug-permeability and transporter assays in Caco-2 and MDCK cell lines. Future Med Chem, 2011. 3(16): p. 2063-77.
45.Smetanova, L., et al., Caco-2 cells and Biopharmaceutics Classification System (BCS) for prediction of transepithelial transport of xenobiotics (model drug: caffeine). Neuro Endocrinol Lett, 2009. 30 Suppl 1: p. 101-5.
46.Administration, U.S.F.a.D., Guidance for Industry: Biopharmaceutics Classification System-Based Biowaivers. 2021, FDA: Silver Spring, MD.
47.Tavelin, S., et al., Applications of epithelial cell culture in studies of drug transport. Methods Mol Biol, 2002. 188: p. 233-72.
48.Wang, Y., et al., Investigation of the Uptake and Transport of Two Novel Camptothecin Derivatives in Caco-2 Cell Monolayers. Molecules, 2022. 27(12).
49.Varma, M.V., O.P. Perumal, and R. Panchagnula, Functional role of P-glycoprotein in limiting peroral drug absorption: optimizing drug delivery. Curr Opin Chem Biol, 2006. 10(4): p. 367-73.
50.SE, E. Caco-2 Permeability Assay – Drug Permeability and Transporters. [cited 2025 July 2, 2025]; Available from: https://www.evotec.com/drug-permeability-and-transporters/caco-2-permeability.
51.Srinivasan, B., et al., TEER measurement techniques for in vitro barrier model systems. J Lab Autom, 2015. 20(2): p. 107-26.
52.Georg Linz, S.D., Lea Steinbeck, Gurbet Köse, Fabian Kiessling, Matthias Wessling, Cell barrier characterization in transwell inserts by electrical impedance spectroscopy. 2022.
53.Marino, A.M., et al., Validation of the 96 well Caco-2 cell culture model for high throughput permeability assessment of discovery compounds. Int J Pharm, 2005. 297(1-2): p. 235-41.
54.Tervonen, A., et al., Structural dynamics of tight junctions modulate the properties of the epithelial barrier. PLoS One, 2019. 14(4): p. e0214876.
55.Otani, T. and M. Furuse, Tight Junction Structure and Function Revisited. Trends Cell Biol, 2020. 30(10): p. 805-817.
56.Lee, B., K.M. Moon, and C.Y. Kim, Tight Junction in the Intestinal Epithelium: Its Association with Diseases and Regulation by Phytochemicals. J Immunol Res, 2018. 2018: p. 2645465.
57.Cui, Y., et al., A Bidirectional Permeability Assay for beyond Rule of 5 Compounds. Pharmaceutics, 2021. 13(8).
58.Kulthong, K., et al., Microfluidic chip for culturing intestinal epithelial cell layers: Characterization and comparison of drug transport between dynamic and static models. Toxicol In Vitro, 2020. 65: p. 104815.
59.Zheng, R., et al., Transport and Permeation Properties of Dapivirine: Understanding Potential Drug-Drug Interactions. Pharmaceutics, 2022. 14(9).
60.Infection, B.H.O.-o.-C.R.I.M.a.M.F.I.S., Bioengineered Human Organ-on-Chip Reveals Intestinal Microenvironment and Mechanical Forces Impacting Shigella Infection. 2019.
61.Gayer, C.P. and M.D. Basson, The effects of mechanical forces on intestinal physiology and pathology. Cell Signal, 2009. 21(8): p. 1237-44.
62.Trietsch, S.J., et al., Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes. Nat Commun, 2017. 8(1): p. 262.
63.Jing, B., et al., Establishment and Application of Peristaltic Human Gut-Vessel Microsystem for Studying Host-Microbial Interaction. Front Bioeng Biotechnol, 2020. 8: p. 272.
64.Zhang, S.Y., et al., Validation of a Caco-2 microfluidic Chip model for predicting intestinal absorption of BCS Class I-IV drugs. Int J Pharm, 2024. 656: p. 124089.