Abstract 11681: Engineering of an IPSC Derived Perfusable and Thick Three-Layer Cardiac Tissue

Introduction: The left ventricular wall is an approximately 1 cm thick tissue composing of vascular networks and three distinct layers: the endocardium, the myocardium, and the epicardium. Engineering of a perfusable and thick three-layer cardiac tissue from human induced pluripotent stem cells (hiPSCs) has not been accomplished to date. Here, we describe formation of a perfusable and thick three-layer engineered cardiac tissue (PTTL-ECT) using additive and subtractive manufacturing. Methods: hiPSCs were differentiated into endocardial (Endo) cells, cardiomyocytes (CMs) and epicardial (Epi) cells. To create a perfusable vascular network in an engineered cardiac tissue, we used a water-soluble heat extruded material to print a sacrificial pattern that would eventually dissolve and become a vascular lumen. By casting hydrogels and the three types of cells derived from hiPSCs, we stacked the endocardial layer, myocardial layer, and epicardial layer mimicking the architecture of the ventricular wall . We then compared PTTL-ECT with engineered tissues without perfusable vascular channels (TTL-ECT) and single layer perfused tissues (PTSL-ECT). Results: Purity of hiPSC derived-Endo cells, -CMs, and -Epi cells reached 96.98±1.33% for NFATC1 + and CD31 + Endo-cells, 83.95±1.77% for cTNT + CMs, and 97.75±1.75% for WT1 + Epi-cells, respectively. The PTTL-ECT had 1.2 cm thickness at day 1 after casting. Histological analysis of the TTL-ECT without vascular channel showed a necrotic core with only the outermost cell layer survived after 4 weeks of culture. Addition of the vascular channel enabled cell survival across the thickness of the entire PTTL-ECT, while the three-layered structure remained intact over four weeks of perfusion culture. CMs in the PTTL-ECT showed enhanced maturity in terms of gene expression profiles, sarcomere lengths and contraction force compared with PTSL-ECT. Conclusions: By combining additive and subtractive manufacturing methods, we successfully generated a perfused, three layers thick cardiac graft that maintained architecture and viability over extended culture periods. The PTTL-ECT could be an useful in vitro model that mimic the native ventricular tissue in disease modeling and drug testing.