Optimized methods for the functional cryopreservation of adult human primary cardiomyocytes

Adult human primary cardiomyocytes (hPCMs) is a high-fidelity and informative cardiac model that is expected to advance our knowledge of the human heart. However, currently, no method exists that recovers hPCMs from cryopreservation with high efficiency, limiting their use as a versatile research model. Based on our previous success with isolated hPCMs, we designed a new strategy that cryopreserves myocardial tissue at a specific step during hPCM dissociation, i.e., after sectioning tissue chunks into tissue slices. This method yielded cell viabilities comparable to that of freshly isolated hPCMs, and surpassed the performance of cell isolation from cryopreserved tissue chunk, as well as recovery of frozen isolated cells. We demonstrate cytoskeletal, ultrastructural, transcriptomic, metabolic, electrophysiological recovery of these cells both over short-term (1 week) and long-term (6-12 months) storage. In addition, these cells responded promptly to external stimuli, including adrenergic stimulation and hypoxic stress, suggesting functional integrity. Our study illustrates an optimized cryopreservation protocol for the functional recovery of cardiomyocytes from myocardial tissue, broadening their applications in basic and translational research, and opens up the possibility for cell banking.