Background Cardiovascular disease remains the leading cause of death worldwide despite significant progress in prevention and treatment. Recently, abnormalities in coronary microcirculation have been observed in patients across the spectrum of cardiovascular diseases. Coronary microvascular dysfunction (CMD) is a heterogeneous condition characterized by increased coronary microvascular resistance and impaired vasodilation. CMD worsens clinical outcomes in leading causes of cardiac mortality such as myocardial ischemia, coronary artery disease, and heart failure, but the mechanism of CMD progression is poorly understood. Two-dimensional culture models of cardiac disease states are limited in applicability to in vivo disease owing to the complex three-dimensional microenvironment of cardiac tissue. Cardiac organoids derived from induced pluripotent stem cells (iPSCs) or primary human cells present an opportunity to better recapitulate the complexity of native myocardium; however, efforts to develop a vascularized, perfusable cardiac spheroid system to study cardiomyocyte-endothelial interactions have thus far been unsuccessful. The objective of this research is to establish a 3D model of vascularized and perfusable myocardium in order to interrogate the onset and progression of CMD.

Methods Tri-lineage cardiac spheroids were developed by aggregating human fibroblasts, endothelial cells transfected with a flow-responsive GFP reporter (KLF2-GFP), and hiPSC-derived cardiomyocytes. A perfusable microvascular niche (MVN) was fabricated by injecting an endothelial and fibroblast-laden fibrin hydrogel into a precast microfluidic chamber. Addition of cardiac spheroids into the hydrogel was termed cardiac spheroids with microvascular network (CS-MVN). The CS-MVN system was cultured for 7 days, after which spheroid morphology, vessel perfusability, and internal hypoxia were assessed by confocal imaging. Vascularization was confirmed by video microscopy and beat-rate analysis during perfusion of the CS-MVN system with isoproterenol.

Results Microscopy of CS-MVNs demonstrate lumenized, perfusable vessels within cardiac spheroids that anastomose with the surrounding microvascular network. Functionally, a microvascular network causes a significantly lower hypoxic burden in the spheroid interior, and vessels in close proximity to the spheroid are less permeable. The CS-MVN system exhibits an immediate chronotropic response to perfused isoproterenol versus a delayed, diffusion-governed response in spheroids seeded in fibrin gels with no microvasculature.

Conclusion We have, for the first time, established a cardiac spheroid featuring complete vascularization and perfusability, enabling advanced in vitro modeling of the cardiac microenvironment and function. Its modular nature will allow interrogation of parameters such as perfused factors, electrophysiology, fluid shear rates, and spatial differences in gene expression when modeling CMD in vitro.