Mechanical function of scaffold-free cardiac tissue fabricated using a temperature-responsive culture dish depends on the degree of shrinkage and load

Cardiac tissues derived from human-induced pluripotent stem cells (hiPSCs) are a promising platform for physiological modeling and drug screening. Among the various strategies used to recreate the<i>in vivo</i>environment for cardiac tissues, mechanical stress has been widely studied for its diverse effects. However, the effects of cellular structure and mechanical loading on the function of scaffold-free tissues remain unclear. Scaffold-free cardiac tissues were fabricated by layering hiPSC-derived cardiomyocytes onto human cardiac fibroblasts in temperature-responsive culture dishes. These tissues were harvested and cultured under fixed tissue lengths (representing the degree of shrinkage) and afterloads (resistance against contraction) in a newly designed culture and measurement device capable of measuring the tensional force under various tissue lengths and afterloads. The contractile force, tissue stiffness, morphology, and gene expression were evaluated. Double-layered cells formed in a ring shape were mounted onto the device in a bundle shape, enabling the measurement of contractile forces generated by spontaneous beating. Additionally, increased contractile force was observed in response to both stretching and<i>β</i>-adrenergic stimulation. The contractile force was influenced by the degree of shrinkage. Tissues set at shorter lengths (greater shrinkage) exhibited significantly reduced force and did not recover by day 7. Additionally, tissues cultured under higher afterloads displayed significantly increased contractile forces and stiffness. Our findings demonstrate that both the initial shrinkage and afterload magnitude critically influence the mechanical function of scaffold-free cardiac tissues. These results highlight the importance of controlling the mechanical environment in scaffold-free tissue engineering.