Abstract The rise of induced pluripotent stem cell (iPSC) technology has revolutionized disease modeling and cardiovascular regeneration. For clinical applications like drug discovery and toxicity screening, organoids must achieve essential complexity—multicellularity, maturation, and functional force generation. While spherical unicellular organoids enable high throughput, their lack of cellular diversity and poor reproducibility limit their relevance for these purposes. More complex multicellular organoids offer greater physiological fidelity but require costly, labor-intensive methods. This presents a major challenge: balancing the need for organoid complexity with the high throughput required for clinical use. This research aims to create reproducible, ring-shaped, multicellular human cardiac organoids that combine structural complexity with functional contractility. The engineered tissues promote cell alignment, enhance physiological relevance, and optimize nutrient diffusion, enabling high-throughput modeling of cardiomyopathies and testing of therapeutic interventions. Ring-shaped, multicellular cardiac organoids were generated from hiPSC-derived cardiomyocytes and cardiac fibroblasts using a step-wise autonomous self-assembly (SASA) protocol, in which fibroblasts were seeded prior to cardiomyocytes to promote extracellular matrix deposition and tissue maturation. Contactless optogenetic pacing controlled contraction frequency, fluorescent staining assessed sarcomere alignment and organization, and video-based motion analysis quantified contraction force and relaxation kinetics. Two organoid engineering strategies were compared: the Step-wise Autonomous Self-assembly (SASA) method and a one-step bicellular seeding approach. The SASA method yielded organoids with significantly improved structural homogeneity, cell survival, and tissue morphology. Sarcomere length was greater in SASA organoids (1.85 µm) than in one-step organoids (1.72 µm, p 0.0006). Moreover, sarcomere alignment was more consistent, with a lower angular dispersion indicating enhanced structural anisotropy (p 0.0001). Under 1 Hz contactless optogenetic pacing, SASA organoids showed higher contraction force (0.431 mN/mm²) compared to one-step organoids (0.136 mN/mm², p 0.0001). To assess tissue relevance, SASA organoids responded to inotropic agents as expected: 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). The initial results demonstrate that the SASA approach, with contactless optogenetic pacing, generates reproducible cardiac organoids with enhanced homogeneity, survival, and contractile function. By balancing complexity and scalability, this platform provides a robust, high-throughput model for drug testing and disease modeling, improving translational relevance.
Moshe et al. (2026) studied this question.