hiPSC-CM Electrophysiology: Impact of Temporal Changes and Study Parameters on Experimental Reproducibility

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) initiative. Despite the considerable benefits hiPSC-CMs provide, concerns surrounding experimental reproducibility have emerged. Our study aimed to investigate the effects of temporal changes and experimental parameters on hiPSC-CM electrophysiology. hiPSC-CMs (iCell cardiomyocyte ² ) were cultured for 14 days and biosignals were acquired using a microelectrode array (MEA) system. Continuous recordings revealed a 22.6% increase in the beating rate and 7.7% decrease in the field potential duration (FPD) during a 20-minute equilibration period. Location specific differences across a multiwell plate were also observed, with hiPSC-CMs in the outer rows beating 8.8 beats per minute (BPM) faster than the inner rows. Cardiac endpoints were also impacted by cell culture duration; from 2-14 days the beating rate decreased (-12.7 BPM), FPD lengthened (+257 ms), and spike amplitude increased (+3.3 mV). Cell culture duration (4-10 days) also impacted hiPSC-CM drug responsiveness (E-4031, nifedipine, isoproterenol). Our study highlights multiple sources of variability that should be considered and addressed 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., culture time, equilibration time, electrical stimulation settings, report raw data values).

We demonstrate that hiPSC-CM electrophysiology measurements are significantly impacted by slight 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 two weeks following defrost.