Scalable production and preclinical validation of human cardiac organoids for myocardial repair

Introduction Cell-based therapies for myocardial infarction (MI) frequently fail clinical translation due to poor cellular engraftment, the risk of post-transplant arrhythmias, and the inherent limitations in large-scale manufacturing and distribution. Cardiac organoids (COs) offer a promising multicellular alternative to single-cell suspensions. Aim Develop a high-yield, scalable platform for human CO manufacturing, and evaluate their efficacy and safety in a clinically relevant swine model of acute MI. Methods COs were derived from human induced pluripotent stem cells in stirred-tank bioreactors, scaling the process from 100 mL to 1 L. To validate cold-chain-free clinical distribution, COs were transported internationally via 24h air freight at room temperature. Twelve immunosuppressed pigs underwent MI induction and were randomized to Short term-CO (8-day follow-up, n=2), Long term-Control (30-day follow-up, n=4), and Long term-CO (30 days, n=6). Thirty minutes post-MI, treated animals received 3500 COs by intramyocardial injections, whereas controls received vehicle. Engraftment was assessed at 8 and 30 days by immunohistofluorescence for human nuclear antigen (HNA). Paracrine effects were evaluated by Picrosirius Red staining and GeoMx spatial transcriptomics. In long-term groups, rhythm was monitored by 15-day ECG Holter recording; cardiac function and scar size by cardiac magnetic resonance (CMR) at days 2 and 29; and electrophysiological properties and arrhythmia inducibility at day 30 using high-density mapping and programmed electrical stimulation. Results Bioreactor scaling increased output to 70000 COs/batch while preserving structure and function. International non-cryogenic transport did not compromise COs. In vivo, HNA⁺ cells were detected in infarct and peri-infarct regions. At 8 days, COs retained spherical architecture, whereas at 30 days, showed successful disaggregation and migration. Spatial transcriptomics revealed reduced pro-fibrotic gene expression in HNA⁺ regions at 30 versus 8 days. Consistently, collagen quantification showed a mild decrease in collagen I content in CO-treated animals compared to controls, although it was statistically not significant. All animals maintained a sinus rhythm without severe ventricular arrhythmias, and electrophysiological testing revealed no group differences, confirming therapy safety. CMR demonstrated significant (P≤0.05) improvements in the Long-term CO group in left ventricular (LV) stroke volume, LV ejection fraction, cardiac output, and cardiac index, along with reduced scar size, compared to controls. Conclusions This platform enables high-yield CO production and simplified logistics by bypassing cryopreservation. Following implantation, COs integrate into the host myocardium without increasing arrhythmia risk, and are associated with improved cardiac function. Larger ongoing studies are expected to further substantiate their therapeutic potential.