Functional isolation, culture and cryopreservation of adult human primary cardiomyocytes

Abstract

Cardiovascular diseases are the most common cause of death globally. Accurately modeling cardiac homeostasis, dysfunction, and drug response lies at the heart of cardiac research. Adult human primary cardiomyocytes (hPCMs) are a promising cellular model, but unstable isolation efficiency and quality, rapid cell death in culture, and unknown response to cryopreservation prevent them from becoming a reliable and flexible in vitro cardiac model. Combing the use of a reversible inhibitor of myosin II ATPase, (-)-blebbistatin (Bleb), and multiple optimization steps of the isolation procedure, we achieved a 2.74-fold increase in cell viability over traditional methods, accompanied by better cellular morphology, minimally perturbed gene expression, intact electrophysiology, and normal neurohormonal signaling. Further optimization of culture conditions established a method that was capable of maintaining optimal cell viability, morphology, and mitochondrial respiration for at least 7 days. Most importantly, we successfully cryopreserved hPCMs, which were structurally, molecularly, and functionally intact after undergoing the freeze-thaw cycle. hPCMs demonstrated greater sensitivity towards a set of cardiotoxic drugs, compared to human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Further dissection of cardiomyocyte drug response at both the population and single-cell transcriptomic level revealed that hPCM responses were more pronouncedly enriched in cardiac function, whereas hiPSC-CMs responses reflected cardiac development. Together, we established a full set of methodologies for the efficient isolation and prolonged maintenance of functional primary adult human cardiomyocytes in vitro, unlocking their potential as a cellular model for cardiovascular research, drug discovery, and safety pharmacology.

Conductive Bacterial Nanocellulose-Polypyrrole Patches Promote Cardiomyocyte Differentiation

“…H9c2 cells have been extensively used in cardiovascular research as an alternative for adult primary cardiomyocytes (PCMs). ,, PCMs are ideally the supreme cellular model for cardiovascular basic research; , however, their use is extremely limited by the technical intricacies in their isolation, culture, and scale production . Despite the latest attempts to optimize PCM isolation, unstable quality and low cell yield are major hurdles that preclude the use of PCMs in nonacute in vitro experiments. , In this scenario, H9c2 surpasses other existing cardiac cell lines (e.g., HL-1, AC16) , and can be further matured toward a cardiomyocyte-like phenotype when treated with retinoic acid (RA). , However, it was brought to our attention that recent studies using H9c2 cells to test distinct biomaterials for cardiac applications did not induce such a cardiomyocyte differentiation, ,− suggesting plausible difficulties to mature H9c2 cells when grown onto engineered substrates. Additionally, other laboratories reported discrepancies when reproducing H9c2 maturation to cardiomyocytes, , indicating some degree of resistance to differentiation.…”

Identification of miRNAs as Biomarkers of Cardiac Protection in Non-Genetically Modified Primary Human Cardiomyocytes Exposed to Halogenated Hypnotics in an In Vitro Model of Transfection and Ischemia/Reperfusion: A New Model in Translational Anesthesia

“…This protocol was developed with primary human cardiomyocytes isolated from normal human ventricular tissue of the adult heart acquired from PromoCell ® ; however, it could be adapted to primary human cardiomyocytes isolated from fresh tissue prepared according to the standard procedures described in several published studies [ 8 ].…”

Isolation of porcine adult cardiomyocytes: Comparison between Langendorff perfusion and tissue slicing-assisted enzyme digestion

“…For Langendorff perfusion, explanted hearts were mounted on a Maquet HL 20 perfusion system, and perfused with our modified Tyrode's solution [3] for 10-15 min to wash out remaining blood. For digestion, oxygenated Ca2+-free Tyrode's solution containing 275 U/ml collagenase type II (Worthingon, LS004176) was maintained at 37˚C, and perfused at a constant rate of 200 ml/min for 1 h. Once the heart became palpably flaccid and pale, it was removed from the perfusion device, and the entire left atrial appendage (LAA), and portions of the right (RV) and left ventricular (LV) free wall were excised, individually triturated with surgical scissors and pipetted repeatedly to disperse cells.…”

“…For TSAD, LAA, RV and LV specimens collected and digested according to our published method with slight modifications [3]. Specifically, we used the University of Wisconsin solution (Belzer, CHD120419) for tissue transport and slicing.…”