Significance Robust and predictive in vitro models of human cardiac tissue function could have transformative impact on our ability to test new drugs and understand cardiac disease. Despite significant effort, the generation of high-fidelity adult-like human cardiac tissue analogs remains challenging. In this paper, we systematically explore the design criteria for pluripotent stem cell-derived engineered cardiac tissue. Parameters such as biomechanical stress during tissue remodeling, input-cell composition, electrical stimulation, and tissue geometry are evaluated. Our results suggest that a specified combination of a 3D matrix-based microenvironment, uniaxial mechanical stress, and mixtures of cardiomyocytes and fibroblasts improves the performance and maturation state of in vitro engineered cardiac tissue.

Aortic valve homeostasis is mediated by valvular interstitial cells (VICs) found in spatially distinct and mechanically dynamic layers of the valve leaflet. Disease progression is associated with the pathological differentiation of VICs to myofibroblasts, but the mechanobiological response profiles of cells specific to different layers in the leaflet remains undefined. Conventional mechanically dynamic macroscale culture technologies require a large number of cells per set of environmental conditions. However, large scale expansion of primary VICs in vitro does not maintain in vivo phenotypes, and hence conventional macroscale techniques are not well-suited to systematically probe response of these cell types to combinatorially manipulated mechanobiological cues. To address this issue, we developed a microfabricated composite material screening array to determine the combined effects of dynamic substrate stretch, soluble cues and matrix proteins on small populations of primary cells. We applied this system to study VICs isolated from distinct layers of the valve leaflet and determined that (1) mechanical stability and cellular adhesion to the engineered composite materials were significantly improved as compared to conventional stretching technologies; (2) VICs demonstrate layer-specific mechanobiological profiles; and (3) mechanical stimulation, matrix proteins and soluble cues produce integrated and distinct responses in layer-specific VIC populations. Strikingly, myofibroblast differentiation was most significantly influenced by cell origin, despite the presence of potent mechanobiological cues such as applied strain and TGF-β1. These results demonstrate that spatially-distinct VIC subpopulations respond differentially to microenvironmental cues, with implications for valve tissue engineering and pathobiology. The developed platform enables rapid identification of biological phenomena arising from systematically manipulating the cellular microenvironment, and may be of utility in screening mechanosensitive cell cultures with applications in drug screening, tissue engineering and fundamental cell biology.

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