From Matrix to Mechanism : Defined microenvironment engineering and Redox-carbonyl stress signaling in Organoid Systems

다국어 초록 (Multilingual Abstract)

This dissertation advances organoid technology along two complementary axes toward reproducible, mechanistically interpretable platforms for modeling tissue physiology and toxicity.
Chapter 1 addresses a central limitation of organoid culture: its dependence on Matrigel, an undefined, batch-variable, and poorly tunable matrix. By engineering a photo-crosslinkable hyaluronic acid–collagen （HA–collagen） hydrogel through riboflavin phosphate–mediated blue-light irradiation, rapid and uniform gelation was achieved with spatiotemporal control over crosslink density. Systematically varying the collagen-to-HA ratio established quantitative structure– function relationships linking matrix composition to stiffness, viscosity, stress relaxation, and pore–fibril architecture. The optimal condition （Col4HA1） reproduced Matrigel-like growth while maintaining a mechanically permissive window that preserved stemness （Lgr5, Sox9）, differentiation （Villin）, and proper E-cadherin/β-catenin–mediated epithelial polarity. Notably, Col4HA1 sustained homeostatic Lgr5 expression throughout passaging, positioning this defined hydrogel as a tunable, reproducible alternative to Matrigel and as a foundation for organ-specific microenvironment design.
Chapter 2 uses murine liver organoids to dissect how carbonyl stress is transduced into hepatocellular injury. The synthetic glucocorticoid dexamethasone （Dex） sensitized hepatocytes to glyoxal （GO）–induced carbonyl stress. GO+Dex co-exposure markedly increased CML accumulation, RAGE expression, and ERK/JNK phosphorylation, accompanied by glutathione （GSH） depletion and a decreased GSH/GSSG ratio. Transcriptomic analysis revealed a UPR-associated signature in which CHAC1 acted as a key effector: GO+Dex elevated Chac1 and Ddit3 expression and enhanced PERK–eIF2α phosphorylation. GSH supplementation and ISRIB blunted these responses and reduced CML accumulation and mitochondrial ROS, with ISRIB additionally lowering RAGE expression and partially restoring respiratory function. These results delineate a mechanism in which glucocorticoid exposure converges with GSH depletion and glyoxal-induced carbonyl stress on a CHAC1-associated PERK–eIF2α integrated stress response, driving redox collapse and mitochondrial injury. Although the engineered hydrogel and the redox-toxicity model were developed in different organoid systems, they constitute two complementary components of a single objective: advancing organoid technology through the design of a defined extracellular microenvironment and the mechanistic dissection of cellular stress responses.
Chapter 1 holds the biological readout constant to define and optimize the matrix, whereas Chapter 2 holds the culture system constant to isolate stress mechanisms, together reflecting a one-variable-at-a-time strategy. The unifying theme is reproducibility: Chapter 1 establishes a chemically defined, organ-tunable microenvironment that removes the batch variability inherent to Matrigel, while Chapter 2 exemplifies the mechanistic redox and toxicity analysis for which such a controlled substrate is ultimately required. Because the present stress model was, of necessity, established in the conventional Matrigel-based system, re-optimizing the defined hydrogel for liver organoids and re-evaluating the GSH/ISR– mitochondrial axis within it represents the logical next step—one through which defined microenvironment engineering and quantitative stress-response readouts can be integrated into a single reproducible platform for disease modeling and toxicity assessment.

목차 (Table of Contents)

• General Introduction 1
• Chapter I. A photo-crosslinkable hyaluronic acid-collagen hydrogel maintains epithelial integrity and supports organoid stemness 5
• Summary 6
• I. Introduction 8
• 1. Organoids as physiological models and the ECM dependency 8
• 1.1 Organoids as advanced in vitro platforms for biomedical research 8
• 1.2 Limitations of Matrigel and the need for defined ECM alternatives 9
• 2. Collagen-Hyaluronic acid composite hydrogels : Desing rationale and engineering principles 10
• 2.1 Biopolymer components: collagen and HA in ECM biomimicry 10
• 2.2 Photo-crosslinking as a spatiotemporally controlled gelation strategy 11
• 3. Intestinal epithelial biology and the mechano-biological framework of organoid stemness 12
• 3.1 Intestinal stem cell niche: LGR5, Wnt/b-catenin, and lineage specification 12
• 3.2 Matrix mechanobiology: how ECM physical properties govern organoid fate 14
• 4. Research purpose 15
• II. Materials and Method 16
• 1. Fabrication of crosslinked hydrogel 16
• 2. Fourier transform infrared （FTIR） spectroscopy 17
• 3. Scanning electron microscopy （SEM) 18
• 4. Rheological characterization 19
• 5. Swelling and weight distribution of hydrogel 20
• 6. Organoid seeding 21
• 7. PI and Calcein AM staining 22
• 8. Immunofluorescence analysis of organoid 23
• 9. Western blotting 24
• 10. Statistical Analysis 25
• III. Result 26
• 1. Distinct crosslinking mechanisms define the mechanical properties of HA-collagen hydrogels 26
• 2. Composite hydrogels integrate structural features of collagen fibrils and HA-derived pores 28
• 3. HA–collagen hydrogels demonstrate enhanced stability and tunable swelling 30
• 4. HA-collagen hydrogels maintain cell viability and support organoid formation 35
• 5. HA-collagen hydrogels support organoid intestinal differentiation and epithelial polarity 37
• 6. HA-collagen hydrogels maintain intestinal characteristics during passages 41
• Ⅳ. Discussion 43
• Chapter II. Dexamethasone exacerbates glyoxal-induced CML/RAGE accumulation and mitochondrial dysfunction via GSH redox imbalance and ISR activation in liver organoids 47
• Summary 48
• I. Introduction 50
• 1. The Liver as a Metabolic and Redox-Challenged Organ 50
• 1.1 Metabolic diversity and oxidative demand in liver 50
• 1.2 Glucocorticoid-induced perturbation of GSH homeostasis 52
• 2. Glyoxal detoxification and carbonyl stress in the liver 54
• 2.1 The glyoxalase–GSH axis as the primary defense against reactive carbonyls 54
• 2.2 AGE-RAGE axis in carbonyl stress-driven cellular damage 56
• 3. Redox imbalance and mitochondrial susceptibility 58
• 3.1 GSH depletion as a sensitizing event in redox buffering failure 58
• 3.2 Mitochondrial dysfunction as a stress amplifier 60
• 4. The Integrated Stress Response and GSH Catabolism 62
• 4.1 The PERK–eIF2α–ATF4–CHAC1 axis in GSH catabolism and hepatocellular injury 62
• 4.2 Chac1-driven GSH dysregulation as feed-forward loop 64
• 5. Research purpose 66
• II. Materials and Method 68
• 1. Organoid seeding 68
• 2. Treatment of liver organoids 72
• 3. Western blotting 73
• 4. RNA sequencing and transcriptomic analysis 74
• 5. Quantitative Real-Time PCR （qPCR) 75
• 6. Mitotracker ＆ MitoSox staining 77
• 7. Oxygen Consumption Rate（OCR） analysis 78
• 8. Statistical Analysis 79
• III. Result 80
• 1. Dexamethasone enhanced glyoxal-induced CML/RAGE-MAPK activation 80
• 2. Dex-induced GSH depletion drives GO-mediated MAPK signaling 82
• 3. GO+Dex induces a UPR-associated transcriptional signature 84
• 4. GO+Dex induces a CHAC1-associated UPR/ISR stress response attenuated by GSH 86
• 5. GSH and ISRIB attenuate GO+Dex-induced CML/RAGE accumulation 88
• 6. ISRIB attenuates GO+Dex-induced mitochondrial oxidative stress and respiratory dysfunction 91
• Ⅳ. Discussion 94
• General Conclusion 99
• References 101
• 국문초록 112