Journal Article

Industrialization of 3D hiPSC-cardiac microtissues for high-throughput cardiac safety and drug discovery screening

Jain, Shushant, Johnson, Benjamin B., Samson-Couterie, Benoit, Mei, Hailiang, Mummery, Christine L., de Korte, Tessa, Bellin, Milena, Yiangou, Loukia, Mol, Mervyn P.H., Kosmidis, Georgios, Davis, Richard P., Razaghi, Ehsan, François, Louise, Meraviglia, Viviana, Braam, Stefan R., van Helden, Ruben W.J., Kuipers, Tom

2026

Overview

An automation-ready workflow enables scalable production and analysis of human pluripotent stem cell–derived 3D cardiac microtissues.The platform supports reproducible, cost-efficient, high-throughput drug screening using calcium transients and voltage signals as functional readouts.Cardiomyocytes from a genetic disease human pluripotent stem cell line, combined with a pharmacological trigger, reliably reproduce an arrhythmic phenotype in the 3D model.Screening more than 2000 compounds confirmed the assay’s ability to identify candidate antiarrhythmic agents.The platform’s advanced technology readiness level supports its use in pharmaceutical testing and regulatory evaluation. Current cardiac cell models for drug screening often face a trade-off between cellular maturity and throughput. 3D human-induced pluripotent stem cell (hiPSC)–based heart models typically exhibit adult-like features, but their use often requires large cell numbers or complex equipment. In this study, we developed cost-effective methods to scale the production of stem cell–derived cardiac microtissues (cMTs) containing three cardiac cell types and assess calcium transients and action potential metrics for high-throughput screening (HTS). Automating the procedure revealed reproducible drug responsiveness and predictive accuracy in a reference compound screen. Furthermore, an arrhythmic phenotype was reliably triggered in cMTs containing cardiomyocytes with an RYR2 mutation. Screening a library of more than 2000 compounds demonstrated the suitability of the assay for identifying potential antiarrhythmic agents. Our findings underscore the scalability of cMTs and their utility in disease modeling and HTS. The advanced technology readiness level of cMTs supports their regulatory uptake and acceptance within the pharmaceutical industry. [Display omitted] 3D cardiac microtissues derived from human pluripotent stem cells offer a physiologically relevant alternative to conventional 2D cultures for cardiac safety and efficacy testing. In this study, we developed a cost-effective, automated platform for high-throughput generation and functional screening of cardiac microtissues. The system enables operator-independent assessment of compound effects on cardiac function using fluorescence-based calcium and voltage assays. With robust detection of both inotropic and arrhythmogenic phenotypes, as well as successful screening of a greater than 2000-compound library, the platform has transitioned from proof-of-concept to a working prototype evaluated under industry-relevant laboratory screening conditions, reaching Technology Readiness Level five. Despite these advances, some challenges remain for full-scale industrial implementation. While the model supports multiplexed readouts, integrating additional end points, such as force measurement or metabolic activity, would further enhance translational relevance but does introduce complexity in assay standardization and data interpretation. In addition, although the system supports the use of patient- and mutation-specific hPSC lines, modeling disease phenotypes remains variable, particularly for conditions with subtle functional changes. To enable broader adoption, standardized criteria for disease phenotype expression and response thresholds will be essential. From a policy standpoint, this work aligns with initiatives such as the FDA Modernization Act 2.0 and the adoption of the ICH S7B/E14 Q&As, both of which promote validated, human-relevant in vitro systems as alternatives to animal testing. Continued alignment with these frameworks, alongside interlaboratory validation, will be critical to transitioning cMTs from research tools to modular, cost-effective, and regulatory-accepted platforms for high-throughput cardiac safety and efficacy assessment. This work establishes scalable, automated methods for producing and screening 3D human stem cell–derived heart tissues. High-throughput screening of more than 2000 compounds shows that this model can predict drug effects on heart rhythm and contractility, paving the way for its use in drug safety and discovery pipelines.

• Publisher: Elsevier Ltd,Elsevier Limited
• Subject: 3D cell models / Anti-Arrhythmia Agents - pharmacology / Antiarrhythmic agents / Automation / cardiac safety / Cardiomyocytes / Cell culture / Drug development / drug discovery / Drug Discovery - methods / Drug Evaluation, Preclinical - methods / Drug screening / Fibroblasts / Genotype & phenotype / Heart / High-Throughput Screening Assays - methods / Human influences / human pluripotent stem cells / Humans / Induced Pluripotent Stem Cells - cytology / Induced Pluripotent Stem Cells - drug effects / Internal Medicine / Labeling / Morphology / Mutation / Myocytes, Cardiac - cytology / Myocytes, Cardiac - drug effects / Pharmaceutical industry / Physiology / Pluripotency / Reproducibility / Ryanodine receptors / Standardization / Stem cells / Technology assessment