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.

心血管疾病是全球最常见的死亡原因。准确模拟心脏稳态、功能障碍和药物反应是心脏研究的核心。成人原代心肌细胞 (hPCMs) 是一种很有前途的细胞模型，但不稳定的分离效率和质量、培养中的快速细胞死亡以及对冷冻保存的未知反应阻碍了它们成为可靠和灵活的体外心脏模型。结合使用肌球蛋白 II ATP 酶的可逆抑制剂 (-)-blebbistatin (Bleb) 和分离过程的多个优化步骤，我们实现了比传统方法提高 2.74 倍的细胞活力，并伴随着更好的细胞形态，微扰的基因表达、完整的电生理学和正常的神经激素信号传导。培养条件的进一步优化建立了一种能够将最佳细胞活力、形态和线粒体呼吸维持至少 7 天的方法。最重要的是，我们成功地冷冻保存了 hPCM，它们在经历冻融循环后在结构、分子和功能上都完好无损。与人类诱导的多能干细胞衍生的心肌细胞 (hiPSC-CMs) 相比，hPCMs 对一组心脏毒性药物表现出更高的敏感性。在群体和单细胞转录组水平上对心肌细胞药物反应的进一步剖析表明，hPCM 反应在心脏功能中更为明显，而 hiPSC-CMs 反应反映了心脏发育。一起，