Our aim is to develop a 3D model unit of cardiac muscle: an in-vitro analog of the trabeculae carneae found in vivo. As a base hydrogel matrix for cardiomyocyte culture in 3D, we develop a blend of decellularized extracellular matrix (dECM) and fibrin. This blend contains essential components of cardiac ECM, provides rapid, cell-friendly coagulation, and closely matches the mechanical properties of native myocardium. Co-culture of the H9c2 model cell line with fibroblast cells in this hydrogel showed enhancement in attachment, spreading, and cardiogenic differentiation of H9c2 cells. This is ascribed primarily to the collagen content of the dECM. Calcium imaging and analysis of beating motion of primary rat neonatal cardiomyocytes cultured in the 3D hydrogel showed specific improvement in recovery, frequency, synchronicity, and beating rates compared to a series of common hydrogel controls, including collagen-fibrin composites. This establishes the dECM-fibrin hydrogel as an optimal base matrix for cardiac tissue culture and engineering. The trabeculae carneae are cardiac muscle fibers at the inner surface of the ventricles. They are an accessible representation of the tiniest building blocks of the cardiac tissue because of their dimensions and cellular orientation. Molding cell-seeded dECM-fibrin hydrogel in microfabricated grooves, we fabricated in vitro analogs of the trabeculae carneae. In these 3D structures, propagation of cell alignment due to the corner contact guidance successfully addresses the challenge of 3D cell orientation. The effect provides alignment 250-300 µm from the corners, enabling full 3D orientation in 350 µm by 350 µm square section microgrooves. The cell-laden hydrogel can be detached from the PDMS surface while maintaining cell alignment. The alignment enhanced the functionality of rat neonatal cardiomyocytes beating by maintaining the contractility of the cells for longer time compared to the random distribution of the cells in the hydrogel. Mechanical forces play key roles in the development and cardiac tissue morphogenesis. Relatively well-known in 2D cultures, knowledge about mechanical effects in 3D is scarcer. We investigate the combined effect of topography and mechanical stimulation on 3D cardiac cell culture. For application of cyclic stretch, we designed and fabricated a user-friendly mechanical stimulator. In 2D cultures, the cells orient perpendicularly to the direction of applied cyclic stretch in agreement with known strain-avoidance mechanisms. In 3D, the cells react to combined topography and mechanical stimulation by adopting an orientation around 45°. This reflects the integration of the conflicting stimuli of alignment along the grooves but perpendicular to the stretch direction. Off-axis alignment may be a novel mechanism for maintenance of helicoidal fiber alignment in the heart. As anticipated, mechanical stimulation also improved the maturation and functionality of the neonatal cardiac cells. Overall, we provide a novel biomaterial for 3D cardiac cell culture and find an effective, yet simple approach to encourage 3D cell alignment. Adding mechanical stretching enhances the maturation and functionality of the bioengineered tissue in vitro, and provides the possibility of off-axis alignment reminiscent of the helicoidal fiber arrangement in the heart. Our trabeculae carneae unit model therefore provides an enhanced 3D environment for investigating cell fate and tissue functionality.