High‐Resolution Imaging and Assessment of Vascular Network in Tri‐Lineage hiPSC‐Derived Cardiac Microtissues

Cardiac microtissues (cMTs) are three‐dimensional, self‐organizing structures that recapitulate key features of native heart tissue. cMTs can be generated using induced pluripotent stem cells (iPSCs) technology by combining iPSC‐derived cardiomyocytes (CMs), endothelial cells (ECs), and cardiac fibroblasts (CFs) in a predefined ratio that closely resembles the cellular composition of the heart. Although cMTs lack true vascular perfusion, the incorporation of endothelial cells promotes the spontaneous formation of microvascular‐like networks, which are identifiable within the 3D structure. The presence of vascular endothelial cells within cMTs enhances cardiomyocyte maturation and contraction force, while also making these models valuable in disease modeling, particularly for conditions involving endothelial dysfunction. High‐resolution imaging of cMTs allows detailed visualization of cellular components and analysis of vascular‐like structures, providing critical insights into the complex interactions between cardiac cells and their microenvironment. Here, we present protocols for the generation and examination of scaffold‐free, vascularized cMTs derived from human iPSCs. First, we outline the steps for aggregation of pre‐differentiated iPSC‐CMs, iPSC‐ECs, and iPSC‐CFs to form vascularized cMTs. We then detail the downstream methodology for fluorescence labeling of both whole‐mount and sectioned cMTs, followed by image acquisition, visualization, and capture using confocal microscopy. Finally, we demonstrate image analysis, and the measurement of the capillary‐like networks using ImageJ (FIJI) software, with 3D modeling and Z ‐stack processing modules. © 2025 Wiley Periodicals LLC. Basic Protocol 1 : Formation of tri‐lineage hiPSC‐derived cardiac microtissues Basic Protocol 2 : Immunofluorescence staining of whole‐mount and sectioned cardiac microtissues Alternate Protocol : Counterstaining of whole‐mount cardiac microtissues for cryo‐sectioning Basic Protocol 3 : Cardiac microtissue imaging setup and data acquisition using confocal microscopy Basic Protocol 4 : Assessment of the vascular network in cardiac microtissues