02766nas a2200337   4500000000100000000000100001008004100002260001500043653002000058653001600078653001800094653003000112100002000142700001400162700001900176700002100195700002000216700002000236700002100256700001800277700001900295700001900314700001800333700001900351700002800370245016000398856005500558300000900613520179200622022001402414       2025                            d  c2025-04-0710aDrug regulation10adrug safety10aLab-on-a-chip10aStem-cell differentiation1 aM. Iveth Garcia1 aKeri Dame1 aVerena Charwat1 aBrian A. Siemons1 aHenrik Finsberg1 aBhavya Bhardwaj1 aRyosuke Yokosawa1 aIshan Goswami1 aDylan Bruckner1 aSamuel T. Wall1 aKevin A. Ford1 aKevin E. Healy1 aAlexandre J. S. Ribeiro00aHuman induced pluripotent stem cell-derived cardiomyocytes and their use in a cardiac organ-on-a-chip to assay electrophysiology, calcium and contractility  uhttps://www.nature.com/articles/s41596-025-01166-4  a1-473 aCardiac organs-on-a-chip (OoCs) or microphysiological systems have the potential to predict cardiac effects of new drug candidates, including unanticipated cardiac outcomes, which are among the main causes for drug attrition. This protocol describes how to prepare and use a cardiac OoC containing cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS cells). The use of cells derived from hiPS cells as reliable sources of human cells from diverse genetic backgrounds also holds great potential, especially when cultured in OoCs that are physiologically relevant culture platforms. To promote the broad adoption of hiPS cell-derived cardiac OoCs in the drug development field, there is a need to first ensure reproducibility in their preparation and use. This protocol aims to provide key information on how to reduce sources of variability during hiPS cell maintenance, differentiation, loading and maturation in OoCs. Variability in these procedures can lead to inconsistent purity after differentiation and variable function between batches of microtissues formed in OoCs. This protocol also focuses on describing the handling and functional assessment of cardiac microtissues using live-cell microscopy approaches to quantify parameters of cellular electrophysiology, calcium transients and contractility. The protocol consists of five stages: (1) thaw and maintain hiPS cells, (2) differentiate hiPS cell cardiomyocytes, (3) load differentiated cells into OoCs, (4) maintain and characterize loaded cells, and (5) evaluate and utilize cardiac OoCs. Execution of the entire protocol takes ~40 days. The required skills to carry out the protocol are experience with sterile techniques, mammalian cell culture and maintaining hiPS cells in a pluripotent state.  a1750-2799