Multi-Organoid Device with Convective Transport for in vivo-like Liver-Pancreas Axis in Obesity

다국어 초록 (Multilingual Abstract)

Multi-Organoid Device with Convective Transport for in vivo- like Liver-Pancreas Axis in Obesity Obesity-related metabolic disorders, including metabolic dysfunction- associated steatotic liver disease (MASLD) and type 2 diabetes mellitus (T2DM), continue to rise, creating an urgent need for in vitro systems that more accurately mimic the physiological communication between the liver and pancreas. Although advancements in multi-organoid culture platforms have improved the ability to study inter-organ interactions, many existing models still fall short of representing the directional and dynamic metabolic exchanges that drive disease progression in vivo. These shortcomings largely stem from passive diffusion–based molecular transfer and the use of uniform media conditions that diminish tissue-specific functions. As a result, conventional systems struggle to reproduce physiologically relevant metabolic responses, limiting their effectiveness in modeling disease progression and therapeutic evaluation. To address these issues, we engineered a multi-organoid device (MOD) that spatially isolates hepatic and pancreatic organoids in distinct media chambers while enabling metabolic exchange through convective flow. Transport efficiency was examined using FITC-dextran, and organoid functionality was assessed by quantifying insulin and albumin secretion. Additional functional assays and transcriptomic profiling were performed to determine the MOD’s capacity to model MASLD-driven diabetic phenotypes. The platform reproduced hallmark features of MASLD-induced T2DM, demonstrating markedly improved directional movement of glucose and other solutes compared with passive diffusion systems, as confirmed by simulations and experimental diffusion analyses. Media compartmentalization helped maintain organoid health and enhanced insulin and albumin production by 1.8- and 1.6-fold relative to non-separated cultures. The system further achieved rapid glucose normalization within two hours of stimulation, approximating physiological glucose handling that has been difficult to achieve in previous in vitro models. Under MASLD-like conditions, liver-secreted Fetuin-A was identified as a contributor to β-cell apoptosis, providing mechanistic insight into the metabolic linkage between hepatic dysfunction and pancreatic failure. Overall, the MOD offers a physiologically relevant and mechanistically informative platform for studying metabolic disease progression and supports future applications in therapeutic discovery. Keyword: multi-organoid device, convective transport, organoids, metabolic dysfunction-associated steatotic liver disease, type 2 diabetes mellitus

목차 (Table of Contents)

• Abstact i
• List of Figures ⅴ
• List of Abbreviations ⅳ
• Chapter 1. Introduction 1
• Chapter 2. Materials and Methods 6
• 2.1. Design and fabrication of the Multi-Organoid Device (MOD) 6
• 2.2. Estimation of wall shear stress in the connecting channel 6
• 2.3. Generation of mouse liver organoids and pancreatic organoids 7
• 2.4. Mouse liver and pancreatic organoid culture 8
• 2.5. Cell culture 10
• 2.6. Quantification of molecular transport and PTFE membrane diffusion10
• 2.7. Measurement of membrane permeability 11
• 2.8. Computational simulation 11
• 2.9. Co-culture of liver and pancreatic organoids in the MOD 12
• 2.10. Palmitate (PA) treatment 13
• 2.11. Quantitative real-time polymerase chain reaction (qRT-PCR) 13
• 2.12. Immunocytochemistry (ICC) staining 14
• 2.13. Cell viability analysis 15
• 2.14. Oil Red O staining 15
• 2.15. Analytical measurements of insulin and Fetuin-A (FetA) secretion . 16
• 2.16. FetA antibody-neutralization assay 16
• iv
• 2.17. Glucose-stimulated insulin secretion (GSIS) analysis 17
• 2.18. In vitro glucose tolerance test (GTT) 17
• 2.19. RNA-sequencing analysis 18
• 2.20. Statistical analysis 19
• Chapter 3. Results 21
• 3.1. Design of a multi-organoid device (MOD) recapitulating the liver–
• pancreas axis 21
• 3.2. Establishment of adult stem cell–derived liver and pancreatic organoids
• . 26
• 3.3. Enhancement of mass transport through convective flow in the MOD29
• 3.4. Cell viability and functional maintenance under separated culture media
• within the MOD 35
• 3.5. Induction of MASLD-like conditions within the MOD 41
• 3.6. MASLD-induced β-cell apoptosis within the MOD 44
• 3.7. Reconstruction of MASLD-driven T2DM phenotypes in the MOD .. 47
• 3.8. Evaluation of Metformin as a Therapeutic Intervention in the MOD . 48
• 3.9. Confirming the FetA-Driven Mechanism of β-Cell Dysfunction in the
• MOD 52
• Chapter 4. Discussion and Conclusion 56
• Reference 60
• 국문초록 67
• v