Engineering vascularization in a heart-on-a-chip system to investigate the effect of intravascular flow on cardiac contraction

Introduction: Cardiac microphysiological systems, small-scale in vitro models of heart tissue, hold great promise for preclinical drug testing and studies of cardiac disease. In particular, cardiac spheroids, aggregates of cell types found in the heart, have the advantage of recapitulating the three-dimensional structure of the myocardium and many of its contractile properties, along with the ability to maintain their function during prolonged culture. However, these spheroids lack vasculature, a key regulator of mechanical and inflammatory signaling and a functional barrier between circulating factors in the blood and heart muscle. Thus, vascularization of cardiac spheroids remains a large technical hurdle that requires development of new engineering approaches. Our strategy to address this need is to integrate cardiac spheroids with self-assembled, perfusable microvascular networks within a microfluidic device with circulating flow. This heart-on-a-chip platform incorporates two important innovations: 1) We can assess the impact of flow on vascular networks using a flow sensor based on the expression of the transcription factor Krüppel-like factor 2 (KLF2), a key regulator of an anti-inflammatory, anti-thrombotic endothelial phenotype. 2) We can control the levels of intravascular flow and assay both cardiac contraction and vascular function in our chip system, allowing us to evaluate the crosstalk between these compartments. Using this heart-on-a-chip platform, we explore vascularization strategies and evaluate the impact of flow on the function of the vascular networks and the cardiac spheroid.

Materials and

Methods: We generated cardiac spheroids by aggregating human induced pluripotent stem cell-derived cardiomyocytes (CM), and primary endothelial cells (EC) and fibroblasts (FB). These cardiac spheroids were suspended in a fibrin gel along with additional EC and FB and injected into our custom microfluidic devices to initiate self-assembly of perfusable microvascular networks around and through the cardiac spheroid, forming a heart-on-a-chip preparation (Fig. 1A). A microfluidic-based pump attached to each chip was used to circulate media through the microvasculature and perfuse the cardiac spheroid (Fig. 1B). Importantly, we generated EC with a fluorescent reporter driven by expression of the flow-sensitive KLF2 promoter (KLF2-GFP EC) and incorporated these cells within the microvascular networks as real-time indicators of endothelial response to flow. To study the impact of intravascular flow on the vasculature, we measured changes in KLF2-GFP expression, evaluated microvascular network barrier function by introducing fluorescent dextran into the vessels and measuring vascular permeability, stained for endothelial cell junction proteins, and measured changes in vascular diameter and branching. To study the impact of intravascular flow on the myocardial compartment, we measured changes in contraction from videos of beating spheroids with and without flow and evaluated response to cardioactive drugs perfused through the vasculature on spheroid contraction parameters. Additionally, we used confocal imaging to assess the degree of cardiac vascularization and perfusion in our preparation and evaluated changes in the expression of EC and CM-associated genes in response to vascularization.

Results, Conclusions, and Discussions: Engineered vessels within our microfluidic device form a fully perfusable network with complex geometry (Fig. 1C). Application of flow activates the KLF2 pathway in the endothelial cells of these vessels, evidenced by increases in GFP fluorescence throughout the networks (Figs. 1C, 1D) and the upregulation of endogenous endothelial KLF2. Further, flow-conditioned vascular networks exhibit increased vascular diameters (Fig. 1E), decreased branching, and decreased permeability (Fig. 1F). Interestingly, the degree of KLF2 expression depends on the local vascular wall shear stress and can be modulated through changes in intravascular flow rates via the pump or by modification of vessel geometry. We demonstrate the integration of cardiac spheroids and microvascular networks and the full vascularization and perfusion of cardiac spheroids (Fig. 1G). Further, we study the effect of intravascular flow parameters, including timing and flow velocity, on spheroid vascularization, spheroid contraction, and KLF2 expression within the spheroid. Finally, we measure changes in CM contraction driven by pharmacological agents that are introduced through the vasculature (Fig. 1H-I).

Altogether, these data indicate that we have developed a fully vascularized and perfusable heart-on-a-chip system in which flow remodels vascular geometry and function and alters cardiac contraction. In doing so, we show how cardiac spheroid composition and vascular flow can be modulated to achieve functional vascularization. This system represents a new platform for dissecting flow-mediated EC-vascular interactions in human models of cardiac function and dysfunction, including the identification of drug effects on both vascular and myocardial function. More broadly, the vascularization approaches defined in these studies could be applied to organoid models of other tissues and to developing organoids to enhance their physiological relevance and to advance their maturation.

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