PROJECT SUMMARY/ABSTRACT Heart disease is the leading cause of death in the U.S., often driven by irreversible cardiac tissue damage leading to heart failure. Cardiac tissue does not naturally regenerate, and thus is an important area of focus for tissue engineering. Previous development into engineered heart tissue (EHT) "patches" with some functional architecture such as pre-patterned vasculature and alignment has shown promising results when implanted into small animal models. Moreover, injected stem cells and implanted cardiac "sheets" have shown some success in restoring some cardiac function when implanted into infarcted hearts of large animals or humans. Despite these successes, scaling EHT that incorporates important functional features such as vascularization and alignment to a physiological thickness (cm-scale) remains a major engineering challenge. The overarching goal of this proposal is to generate in vitro physiologically thick heart tissue patches that can ultimately be implanted into patients to replace damaged tissue. Two key considerations for recapitulating native tissue are (1) cardiac tissue is highly vascularized, and (2) alignment of cells and extracellular matrix within each physiological layer is critical to function. We address these challenges utilizing novel open microfluidic patterning approaches. Our open microfluidic technological advancement offers unique benefits to EHT; for example, it is compatible with virtually any hydrogel, including standard extracellular matrix material such as collagen and fibrin, used extensively for EHT. It is also compatible with specialized stimuli-responsive engineered hydrogels, opening up possibilities for complex engineered tissues with spatial and temporal control. Further, the flow of precursor fluid is driven by passive surface tension forces; thus, sensitive stem-cell- derived cells are not exposed to shear stress from extrusion through a needle or photochemical crosslinking, which are requirements for other tissue fabrication techniques such as 3D bioprinting. Finally, a large area (cm- scale) can be patterned with a single pipetting step, making this fabrication approach ideal for generating large (cm-scale) tissues. In this proposal, I apply these unique attributes of open microfluidic pattering to EHT. Specifically, the ability to pattern enzymatically degradable gels through a background of standard cell culture ECM materials such as collagen or fibrin enables the patterning of complex vasculature in three dimensions. I will also take advantage of previously demonstrated modular stacking of open microfluidic devices and suspended microfluidics to generate aligned EHT patches. In these patches, the tissue is anchored on either end, inducing ECM remodeling and alignment. Each layer is generated and aligned independently. Then, they are stacked together at an angle from the previous layer, creating a multilayered tissue mimicking the heart's helical tissue fiber alignment. As...