The influence of media on toxicity testing using human iPSC-derived cardiomyocytes in monoculture and co-culture models

Abstract

The FDA Modernization Act 2.0 underscores the urgent need for non-animal-based new approach methodologies (NAMs) for chemical safety assessment. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are emerging as critical tools in in vitro cardiotoxicity studies, with iPSC-derived cardiac fibroblasts and endothelial cells enabling the development of advanced co-culture and 3D cardiac models. However, serum-free, off-the-shelf media that support both acute and chronic toxicity testing across a broad pharmacological range remain scarce. To address this, we developed a suite of cell culture media and supplements, including iCell Cardiomyocytes Serum-Free Medium (iCSFM), iCell CardioTox Assay Medium (iCTAM), and the iCell Cardiac Co-Culture Supplements. These were specifically designed to maintain cardiomyocyte function, enable flexibility across assay platforms, and support reproducible toxicity testing in 2D (monoculture) and 3D (co-culture) formats. Cardiac function was assessed using multiple platforms: microelectrode array (MEA), voltage-sensitive dye (VSD), and calcium flux assays in 2D monoculture. For toxicity testing, standard viability endpoints were employed using iCSFM and iCTAM to evaluate compounds with known cardiotoxic effects. Seahorse XF (metabolism) and impedance assays (contractility) assessed metabolic and functional toxicity profiles. Compatibility with transduction methods (AAV, BacMam) and hypertrophy induction (ET-1) was also tested. Additionally, functional toxicity was further examined through 3D co-cultures incorporating iPSC-CM, cardiac fibroblasts, and endothelial cells. MEA results demonstrated consistent field potential durations (FPDs), with long-term culture in iCSFM showing slight FPD shortening, while enhancing assay reliability for albumin-binding compounds. A panel of hERG blockers (E-4031, dofetilide, ondansetron) showed dose-dependent effects on cardiac action potential morphology. Metabolic assays revealed decreased maximal respiration with cardiotoxic molecules, while cardiac contractility decreased significantly with chronic idarubicin exposure and increased contraction amplitudes with (S)-BayK-8644. The incorporation of 3D co-culture models showed improved biological relevance, capturing positive inotropic responses and immune response toxicology. These findings underscore the utility of human iPSC-CM for in vitro cardiac function and cardiotoxicity assessment. Our optimized serum-free media formulations enable robust performance in diverse assay platforms, offering a human-relevant approach for pharmacological and toxicological evaluations. These innovations align with regulatory goals to advance NAMs and reduce reliance on animal testing.