Structural and functional benchmarking of monolayer- and bioreactor-generated hiPSC-derived cardiomyocytes

Transitioning from animal to human cell sources represents a critical milestone in cardiac tissue engineering and biomedical research. Neonatal rat ventricular myocytes (NRVMs) have long served as the functional benchmark for engineered cardiac tissues; however, their rodent origin limits clinical relevance. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer a renewable, species-specific alternative but remain restricted by immature structure and function, small-scale yield, and high batch variability by conventional two-dimensional monolayer (2D-Mono) differentiation. Here, we systematically evaluated hiPSC-CMs generated by 2D-Mono and three-dimensional embryoid-body (3D-EB) differentiation using identical 15-day Wnt-modulated protocols without additional maturation steps. The 3D-EB method yielded 181 × 10⁶ cells per 100 ml, approximately 2.7-fold higher than the 2D-Mono, while maintaining >80% cTnT⁺ purity and reduced batch variability. Structural analyses revealed improved sarcomeric organization in 3D-EB tissues compared with 2D-Mono, although both remained less organized than NRVMs, while other morphological parameters were comparable between groups. Functionally, 3D-EB tissues exhibited faster calcium conduction (33.7 cm/s), indicating enhanced electrical coupling relative to 2D-Mono. Although contractile performance remained similar between differentiation formats and below NRVM levels, 3D-EB tissues exhibited consistent structural and functional improvement in calcium wave velocity and contractility over time. These results show that, even without external maturation cues, 3D-EB differentiation yields reproducible, scalable, and human-relevant cardiomyocytes.