Animal-Origin-Free Method for Generating Blood Vessel Organoids

BackgroundBlood 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. MethodsWe 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. ResultsBVO 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 PDGFR{beta} 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. ConclusionOur 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. HighlightsO_LIEntire blood vessel organoid (BVO) workflow performed in a single ultra-low attachment-96 plate C_LIO_LIFully animal-origin-free: no Matrigel or Geltrex required throughout the protocol C_LIO_LIRobust generation of BVOs using human collagen-based ECM C_LIO_LIHigh-throughput compatible and automation-ready "sitting drop" culture system C_LIO_LIIntegration of BVO-derived cells into host vessels in vivo C_LI