PROJECT SUMMARY Ischemic heart disease accounts for approximately 42.5% of all cardiovascular-related deaths and afflicts 720,000 individuals/year. While current medical treatments have significantly decreased heart attack- associated mortality, post-ischemic myocardial tissue undergoes pathologic remodeling and scarring, subjecting patients to cardiac dysfunction and heart failure. As endogenous cardiac regeneration is limited, stem cell and regenerative medicine-based approaches to cardiac repair represent promising solutions towards regaining normal heart function. Bolstering interest in cardiovascular tissue engineering has given rise to the development of novel biomaterials capable of maintaining cardiac cell function within a 3D tissue-like environment, while detailed protocols have been developed to derive mature cardiomyocytes from a myriad of stem cell sources; however, the essential ability to generate a construct that recapitulates the cellular density and composition, thickness, and helicoid structure of the native heart has limited the therapeutic applicability of tissue engineered constructs thus far. Utilizing photodegradable polymer-based hydrogels that enable a spatially-defined co-culture of hES-derived cardiomyocytes and endothelial cells, we propose to generate a densely-vascularized 3D cardiac construct exhibiting biomimetic helical tissue architecture. To support the culture of a thick myocardial tissue and enhance mass transport of oxygen and nutrients, vessels will be photopatterned into cell-laden hydrogels containing mature cardiomyocytes to generate perfusable vasculature with similar size, shape, and structure to that of the native heart. Channels will be endothelialized and perfused with fresh media using biomimetic pulsatile flow to encourage endothelial-cardiomyocyte cell interaction. We aim to demonstrate that by controlling micron-scale channel architecture in a biomimetic helical arrangement with near-native heart capillary density, paracrine signaling, ECM deposition, and direct contact of endothelial cells and cardiomyocytes, we can direct cardiomyocyte orientation, enhance construct contractility, and recapitulate the native torsional tissue contraction. As the proposed research will represent the first successful strategy to generate perfusable vasculature networks with 3D features on the single-micron scale, we expect that the developed methodologies will find wide applicability in the engineering of vascularized constructs beyond cardiac tissue.