Animal-origin-free method for generating blood vessel organoids

Blood vessel organoids (BVOs) represent a promising tool for modeling vascular diseases, drug screening, and regenerative therapies. However, current protocols for BVO generation are complex, labor-intensive, and reliant on animal-derived extracellular matrices (ECM) such as Matrigel, limiting reproducibility, scalability, and clinical applicability. We developed a simplified, animal-origin-free protocol for BVO generation that addresses current limitations and enables high-throughput automated workflows. The method employs ultra-low attachment 96-well U-bottom plates for standardized aggregation and differentiation of human induced pluripotent stem cells (hiPSCs) in a human derived collagen-based extracellular matrix. Unlike conventional protocols where aggregates are embedded in a two-layer ECM, our approach utilizes a single-layer, which we termed "sitting drop". This innovative approach requires considerably fewer materials and handling steps and is compatible with high-throughput automated machines. BVO generation utilizing the here described optimized protocol resulted in the formation of BVOs with reproducible morphology and cellular composition. Flow cytometry confirmed the presence of CD31⁺ endothelial cells and PDGFRβ⁺ pericytes in BVOs, generated in sitting drops in ultra-low adhesive U-bottom shaped 96 well plates, with cell population percentages comparable to those observed in traditional two-layer BVO cultures. In vivo transplantation of mature BVOs in a mouse full-thickness skin wound model demonstrated integration of BVO derived cells into host vessels, highlighting their potential in cell-based therapies. Our study presents a robust and animal-origin-free method for BVO generation based on single-layer "sitting drop" cultures. This protocol maintains cellular integrity while enhancing reproducibility and automation-readiness, paving the way for high-throughput screening and clinical translation of vascular organoid technology.