Nanopatterned Cell Sheet Assembly of Biomimetic Cardiac Laminae for Modeling Structure-Function Relationships

Replicating the intricate 3-dimensional architecture and coordinated function of native human myocardium remains a central challenge in cardiac tissue engineering. Here, we present a scaffold-free strategy to fabricate multilayered human cardiac tissues with tunable structural anisotropy and physiologically relevant helical alignment. By integrating biomimetic nanotopographical patterning with a thermoresponsive polymer interface, we generated aligned cardiac cell sheets that could be detached and transferred intact. To ensure robust sheet formation and release, our comprehensive investigation found that a coculture system incorporating human induced pluripotent stem cell-derived endocardial-like endothelial cells was essential for facilitating extracellular matrix deposition and maintaining tissue integrity during detachment, outperforming coculture conditions using other stromal cell types. A glycidyl methacrylate (GMA)-modified polyurethane acrylate substrate functionalized with poly(N-isopropylacrylamide) enabled temperature-controlled release, with 0.5% GMA yielding optimal performance. Stacked cardiac sheets with defined angular offsets were used to engineer 4-layered laminae that mimicked the transmural fiber orientation of the ventricular wall. These helically aligned tissues exhibited enhanced contractile synchrony and superior contractile function compared to unaligned or unpatterned controls, as quantified by vector-based contraction analysis. This work introduces a modular, bottom-up platform for constructing functionally anisotropic cardiac tissues, providing new tools for probing myocardial biomechanics, studying development and disease, and informing regenerative therapies.