Mechanical stimulation enhances development of scaffold-free, 3D-printed, engineered heart tissue grafts

Current efforts to engineer a clinically relevant tissue graft from human-induced pluripotent stem cells (hiPSCs) have relied on the addition or utilization of external scaffolding material. However, any imbalance in the interactions between embedded cells and their surroundings may hinder the success of the resulting tissue graft. Therefore, the goal of our study was to create scaffold-free, 3D-printed cardiac tissue grafts from hiPSC-derived cardiomyocytes (CMs), and to evaluate whether or not mechanical stimulation would result in improved graft maturation. To explore this, we used a 3D bioprinter to produce scaffold-free cardiac tissue grafts from hiPSC-derived CM cell spheroids. Static mechanical stretching of these grafts significantly increased sarcomere length compared to unstimulated free-floating tissues, as determined by immunofluorescent image analysis. Stretched tissue was found to have decreased elastic modulus, increased maximal contractile force, and increased alignment of formed extracellular matrix, as expected in a functionally maturing tissue graft. Additionally, stretched tissues had upregulated expression of cardiac-specific gene transcripts, consistent with increased cardiac-like cellular identity. Finally, analysis of extracellular matrix organization in stretched grafts suggests improved remodeling by embedded cardiac fibroblasts. Taken together, our results suggest that mechanical stretching stimulates hiPSC-derived CMs in a 3D-printed, scaffold-free tissue graft to develop mature cardiac material structuring and cellular fates. Our work highlights the critical role of mechanical conditioning as an important engineering strategy toward developing clinically applicable, scaffold-free human cardiac tissue grafts.