Abstract：
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are frequently used for preclinical cardiotoxicity testing and remain an important tool for confirming model-based predictions of drug effects in accordance with the comprehensive in vitro proarrhythmia assay (CiPA). Despite the considerable benefits hiPSC-CMs provide, concerns surrounding experimental reproducibility have emerged. We investigated the effects of temporal changes and experimental parameters on hiPSC-CM electrophysiology. iCell cardiomyocytes2 were cultured and biosignals were acquired using a microelectrode array (MEA) system (2-14 days). Continuous recordings revealed a 22.6% increase in the beating rate and 7.7% decrease in the field potential duration (FPD) during a 20-min equilibration period. Location-specific differences across a multiwell plate were also observed, with iCell cardiomyocytes2 in the outer rows beating 8.8 beats/min faster than the inner rows. Cardiac endpoints were also impacted by cell culture duration; from 2 to 14 days, the beating rate decreased (-12.7 beats/min), FPD lengthened (+257 ms), and spike amplitude increased (+3.3 mV). Cell culture duration (4-10 days) also impacted cardiomyocyte drug responsiveness (E-4031, nifedipine, isoproterenol). qRT-PCR results suggest that daily variations in cardiac metrics may be linked to the continued maturation of hiPSC-CMs in culture (2-30 days). Daily experiments were also repeated using a second cell line (Cor.4U). Collectively, our study highlights multiple sources of variability to consider and address when performing hiPSC-CM MEA studies. To improve reproducibility and data interpretation, MEA-based studies should establish a standardized protocol and report key experimental conditions (e.g., cell line, culture time, equilibration time, electrical stimulation settings, and raw data values).NEW & NOTEWORTHY We demonstrate that iCell cardiomyocytes2 electrophysiology measurements are impacted by deviations in experimental techniques including electrical stimulation protocols, equilibration time, well-to-well variability, and length of hiPSC-CM culture. Furthermore, our results indicate that hiPSC-CM drug responsiveness changes within the first 2 wk following defrost.
摘要：
人类诱导的多能干细胞衍生的心肌细胞（hiPSC-CM）经常用于临床前心脏毒性测试，并且仍然是根据综合体外致心律失常试验（CiPA）确认基于模型的药物作用预测的重要工具。尽管hiPSC-CM提供了相当大的好处，围绕实验可重复性的担忧已经出现。我们研究了时间变化和实验参数对hiPSC-CM电生理的影响。培养iCell心肌细胞2,并使用微电极阵列(MEA)系统获得生物信号(1-14天)。连续记录显示，在20分钟的平衡期间，跳动率增加了22.6％，场电位持续时间（FPD）减少了7.7％。还观察到整个多孔板的位置特定差异，外排的iCell心肌细胞2比内排的每分钟（BPM）快8.8次。心脏终点也受细胞培养时间的影响；从2-14天开始，搏动率降低（-12.7BPM），FPD加长(+257ms),和尖峰幅度增加（+3.3mV）。细胞培养时间（4-10天）也影响心肌细胞药物反应性（E-4031，硝苯地平，异丙肾上腺素）。qRT-PCR结果表明，心脏指标的每日变化可能与培养物中hiPSC-CM的持续成熟（2-30天）有关。还使用第二细胞系(Cor.4U)重复每日实验。总的来说，我们的研究强调了在进行hiPSC-CMMEA研究时需要考虑和解决的多种变异性来源.为了提高可重复性和数据解释，基于MEA的研究应建立标准化的方案并报告关键的实验条件(例如，细胞系,文化时间,平衡时间,电刺激设置,原始数据值)。
