Novel 2D/3D Hybrid Organoid System for High-Throughput Drug Screening in iPSC Cardiomyocytes

Abstract Human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) allow for high-throughput evaluation of cardiomyocyte (CM) physiology in health and disease. While multimodality testing provides a large breadth of information related to electrophysiology, contractility, and intracellular signaling in small populations of iPSC-CMs, current technologies for analyzing these parameters are expensive and resource-intensive. We sought to design a 2D/3D hybrid organoid system and harness optical imaging techniques to assess electromechanical properties, calcium dynamics, and signal propagation across CMs in a high-throughput manner. We validated our methods using a doxorubicin-based system, as the drug has well-characterized cardiotoxic, pro-arrhythmic effects. hiPSCs were differentiated into CMs, assembled into organoids, and thereafter treated with doxorubicin. The organoids were then replated to form a hybrid 2D/3D iPSC-CM construct where the 3D cardiac organoids acted as the source of electromechanical activity which propagated outwards into a 2D iPSC-CM sheet. The organoid recapitulated cardiac structure and connectivity, while 2D CMs facilitated analysis at an individual cellular level which recreated numerous doxorubicin-induced electrophysiologic and propagation abnormalities. Thus, we have developed a novel 2D/3D hybrid organoid model that employs an integrated optical analysis platform to provide a reliable high-throughput method for studying cardiotoxicity, providing valuable data on calcium, contractility, and signal propagation.