Generation of Chamber-Specific 3D Cardiac Organoids and an Integrated Electro-Mechanical Optical Analysis Platform

다국어 초록 (Multilingual Abstract)

Cardiovascular disease remains a leading cause of global mortality, yet the development of effective therapeutics is hindered by the limited predictive accuracy of conventional preclinical models. Human induced pluripotent stem cell-derived cardiomyocytes （hiPSC-CMs） offer a physiologically relevant alternative, and three- dimensional （3D） culture further improves structural maturity over 2D monolayers. However, many generic 3D approaches still fail to recapitulate the chamber-specific electrophysiology of the native human heart, and a standardized platform for objectively quantifying the multi-modal functional output of engineered cardiac tissues is lacking. To address these gaps, we used a biphasic differentiation protocol with stage-specific retinoic acid （RA） modulation to generate chamber-specific 3D cardiac organoids — atrial （aCOs） and ventricular （vCOs）. We developed iCAMAnalyzer, an integrated, Python-based platform that unifies intracellular Ca²⁺ transient kinetics, spatiotemporal activation mapping, and label-free mechanical contractility within a single graphical interface. The activation-mapping module was validated against synthetic ground-truth datasets, showing near-ideal recovery of conduction-velocity metrics （R² 〉 0.99）, with propagation patterns cross-confirmed by independent frame-by-frame （ImageJ） analysis. Morphological and molecular assessment confirmed chamber-specific maturation: vCOs developed longer sarcomeres （~2.1 µm） with MLC2v and Cx43, whereas aCOs showed shorter sarcomeres （~1.7 µm） with MLC2a and Cx40. Using iCAMAnalyzer, we established quantitative ＂functional fingerprints＂ via Shape and Efficiency Indices: vCOs produced plateau-shaped Ca²⁺ transients （high Shape Index）, whereas aCOs produced rapid, spike-shaped transients （low Shape Index, high Efficiency Index）. Physiological fidelity was further supported by chamber-selective pharmacology — for example, carbachol elicited a selective negative chronotropic effect in aCOs. Finally, compatibility with a 3D-printed micro-pillar array demonstrated scalability toward high-throughput screening. In conclusion, this integrated optical platform for electro- mechanical analysis provides a robust, objective, and physiologically relevant framework for precision cardiotoxicity screening and patient-specific disease modeling.

목차 (Table of Contents)

• 1. Introduction 10
• 2. Literature Review 12
• 2.1 Historical Milestones in Cardiac Tissue Engineering 12
• 2.2 Paradigm Shift: From 2D Cultures to 3D Chamber-Specific Models 14
• 2.3 Structural and Functional Differences Between Atrial and Ventricular Tissues 17
• 2.4 Translating Chamber-Specific Principles into hiPSC-Derived Models 20
• 2.5 The Evolution of Functional Quantification Tools: The Necessity for a Digital Analytical Platform 27
• 3. Materials and Methods 29
• 3.1 Human iPSC Culture and 2D (Monolayer) Cardiac Differentiation 29
• 3.2 Generation of Chamber-Specific 3D Cardiac Organoids 29
• 3.3 Fabrication of 3D-Printed Micro-pillar Arrays and Pacing Platform 31
• 3.4 Immunofluorescence Staining 33
• 3.5 Gene Expression Analysis by qRT-PCR 33
• 3.6 Flow Cytometry for Cardiomyocyte Purity 34
• 3.7 Optical Imaging for Ca²⁺ Transient Recording 34
• 3.8 iCAMAnalyzer: A Unified Python Platform for Ca²⁺ and Motion Analysis 35
• 3.9 Pharmacological Treatment 43
• 3.10 Cell Viability Assessment via Live/Dead Assay 43
• 3.11 Statistical Analysis 44
• 4. Results 47
• 4.1 An Integrated Optical Platform for Electro-Mechanical Analysis 47
• 4.2 Efficient Generation and Validation of Chamber-Specific Cardiomyocytes 47
• 4.3 Biphasic Differentiation and Morphological Evolution of 3D Cardiac Organoids 51
• 4.4 Morphological Characterization and Sarcomeric Maturation 53
• 4.5 Molecular Identity and Gap Junction Networking 55
• 4.6 Dynamic Calcium Transient Profiling and Functional Fingerprinting 57
• 4.7 Spatiotemporal Conduction and Electrical Pacing Fidelity 60
• 4.8 Label-Free Mechanical Contractility 63
• 4.9 Chamber-Specific Pharmacological Validation 66
• 4.10 Proof-of-Concept High-Throughput Multi-Organoid Platform 71
• 5. Discussion 74
• 5.1 Enhanced Structural and Functional Maturation in 3D Environments 74
• 5.2 Preservation of Chamber-Specific Identity via Timely Retinoic Acid Modulation 74
• 5.3 Parallel Quantification and Pharmacological Fingerprinting via iCAMAnalyzer 75
• 5.4 Limitations and Technical Constraints 75
• 5.5 Future Directions 76
• 6. Conclusion 78
• 7. References 79