Human-induced pluripotent stem cells (hiPSCs) have revolutionized the world of basic and translational research. In particular, hiPSCs can be differentiated into cardiomyocytes (hiPSC-CMs), emerging as a reference system for cardiac studies due to their biological similarity to primary CMs (Gisone et al., 2022). However, compared to adult ventricular CMs, the main limitation of hiPSC-CMs is their phenotypic immaturity (Denning et al., 2016). To overcome this limitation and to reproduce the in vivo heart microenvironment better, researchers proposed the co-culture of hiPSC-CMs with other cardiac cells, such as endothelial cells (ECs) and fibroblasts (Giacomelli et al., 2020; Campostrini et al., 2021). Besides the influence of cell composition, it is also established that three-dimensional (3D) in vitro cell cultures can better mimic the tissue environment of cells in the organism, preserving cells’ morphology, phenotype, and polarity, thus allowing for a better study of cells’ physiological function and, in turn, obtaining a reliable tissue response (Gisone et al., 2022). This work presents the results of the characterization of hiPSC-CMs + Human Coronary Artery Endothelial Cells (HCAECs) co-culture in a 2D and 3D environment as a cardiac tissue replica for pathophysiological and/or toxicological studies.