A 96-well platform for blood vessel organoid construction

Cardiovascular diseases remain a leading cause of mortality and are closely associated with vascular dysfunction. Blood vessel organoids (BVOs) derived from human pluripotent stem cells provide a three-dimensional model for studying vascular development and disease. Here, we adapted the differentiation framework of Wimmer et al. to a U-bottom 96-well ultra-low-attachment format. Equal numbers of human pluripotent stem cells were seeded into separate wells and differentiated to BVOs. The one-aggregate-per-well configuration prevents fusion between neighboring aggregates and enables each BVO to be cultured, treated, sampled, collected, and analyzed independently. We generated BVOs from H9 embryonic stem cells and induced pluripotent stem cells and assessed vascular marker expression and organization by RT-qPCR and immunofluorescence. Different wells can be assigned to distinct cell lines, treatment groups, doses, conditions, or time points, while plate occupancy can be adjusted to the experimental design. These features provide a scalable standard-plate platform for higher-throughput and multiplexed BVO studies and a basis for future compound-testing applications.