(1188-A) Optimizing culture conditions for cardiac organoids to enhance their health and minimize necrotic patterns

Abstract: The evaluation of cardiotoxicity induced by genetic abnormalities and drug exposure is not well elucidated. Two-dimensional (2D) in vitro culture formats fail to fully recapitulate the 3D tissue microenvironment, while in vivo animal models fail to capture the human-specific responses elicited from drug therapies. The advancement of cardiac differentiation techniques on human induced pluripotent stem cells (hiPSCs) have enabled hiPSCs to serve as a source for the formation of cardiac organoids, which can better predict patient-specific cardiotoxicity due to their enhanced emulation of molecular and cellular interactions. However, optimization of ideal cardiac organoid models that can be generated robustly for higher-throughput drug screening purposes is not well understood. We studied self-assembled cardiac organoids seeded at two seeding densities - 20,000 and 80,000 cells per organoid - (to generate organoids with diameters of 400 µm and 800 µm, respectively by Day 2) and examined their health by looking at the levels of necrosis and cellular composition of these organoids, and maintenance of the organoid’s spherical morphology over fourteen days. To examine the functionality across this time, we evaluated the metabolic activity, degree of fibrosis, and beating kinetics of organoids culture. While the size of the organoids stabilized at 400 µm, regardless of seeding density within seven days, we noticed several differences in the overall health of the organoids. We found that organoids seeded at 20,000 cells per organoid showed reduced levels of necrosis, as evaluated from cleaved caspase 3 staining. We also found organoids seeded at this lower density to be more metabolically active, even during prolonged culture of 14 days, by examining mitochondrial activities using high resolution imaging modalities. Looking at gene expression, we found organoids to be the healthiest at Day 7. Taken together, we propose that organoids seeded at 20,000 cells per organoid and cultured for seven days to be the most optimized culture technique of hiPSC-derived cardiac organoids for drug screening purposes. To enable higher-throughput drug screening evaluation, we introduced our cardiac organoids onto our developed OrganoidChip for immobilization and long-term culture to allow for high resolution monitoring across time. Together, we hope to help develop techniques that capture relevant biological processes on culture platforms that can be monitored in larger scale, automated formats for the evaluation of drug-induced cardiotoxicity.