A novel stepwise autonomous guided assembly approach for engineered cardiac organoid formation

Background The rise of induced pluripotent stem cell (iPSC) technology has revolutionized disease modeling and cardiovascular regeneration. For clinical applications such as drug discovery and toxicity screening, organoids must achieve multicellularity, maturation, and functional force generation. Current organoid platforms can enable high throughput; however, limited cellular diversity and reproducibility restrict translational relevance. More complex multicellular systems provide greater physiological fidelity but require costly and labor-intensive protocols, creating a fundamental challenge between complexity and scalability. Purpose Here, we present SAGA (Stepwise Autonomous Guided Assembly), a paradigm shift in cardiac tissue engineering that leverages temporal cellular organization to generate structurally and functionally superior organoids autonomously. Methods Ring-shaped multicellular human cardiac organoids were generated from hiPSC-derived cardiomyocytes and cardiac fibroblasts using the SAGA method, in which cardiac fibroblasts are seeded prior to cardiomyocytes. This sequential seeding enables fibroblast-driven deposition of authentic extracellular matrix components, including fibronectin, before cardiomyocyte integration, thereby promoting tissue maturation and structural organization. Contactless optogenetic pacing controlled contraction frequency, fluorescent staining assessed sarcomere alignment and organization, and video-based motion analysis quantified contraction force and relaxation kinetics. Results SAGA was compared to a one-step bicellular seeding approach. Sarcomere length was greater in SAGA organoids (1.85 µm) than in one-step organoids (1.72 µm, p < 0.0006), with lower angular dispersion indicating enhanced anisotropy (p < 0.0001). Under 1 Hz contactless optogenetic pacing, SAGA organoids generated higher contraction force (0.380 mN/mm²) compared to one-step organoids (0.141 mN/mm², p < 0.0001). SAGA organoids also demonstrated appropriate pharmacological responsiveness: 0.5 µM Isoproterenol increased beating frequency (~0.9 → 1.3 Hz) and contraction stress (0.594 → 0.841 mN/mm², p < 0.0001), while 0.9 µM Mavacamten reduced contraction stress (0.606 → 0.300 mN/mm², p < 0.0001). Conclusion These findings establish SAGA as a scalable and reproducible engineering strategy that overcomes the long-standing trade-off between complexity and throughput, redefining cardiac organoid fabrication through biologically guided ECM-first assembly.