Animal-Origin-Free Method for Generating Blood Vessel Organoids

Background 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. Methods 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. Results 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, 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 successful integration of BVO derived cells into host vessels, highlighting their potential in cell-based therapies. Conclusion 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. Highlights Entire blood vessel organoid (BVO) workflow performed in a single ultra-low attachment-96 plate Fully animal-origin-free: no Matrigel or Geltrex required throughout the protocol Robust generation of BVOs using human collagen-based ECM High-throughput compatible and automation-ready “sitting drop” culture system Integration of BVO-derived cells into host vessels in vivo