Abstract
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.
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The data, presented in this study, are available from the corresponding author on reasonable request.
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Acknowledgements
The authors would like to thank Sophia Wedel and Maria Dumitrascuta for their excellent preliminary work that formed the basis of this manuscript. We further acknowledge the Tyrolean Cancer Research Institute (TKFI) for their generous support and for providing access to their facilities and equipment.
Funding
Standortagentur Tirol, Health Hub grant, TAM: TM-12514133. Austrian Research Promotion Agency (FFG), Project Number: FO999925858. European HORIZON, Grant agreement ID: 101135053.
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A.H as first author designed and performed experiments, carried out the statistical analyses and wrote the manuscript. D.S. and J.T. designed and performed experiments and contributed to the writing of the manuscript. Y.H.T. performed image analysis. J.F.B. contributed to the mouse experiment. T.E.Y. initiated and supervised the project, designed experiments and wrote the manuscript as corresponding author.
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Hoffmann, A., Schorn, D., Thönig, J. et al. Animal-origin-free method for generating blood vessel organoids. Sci Rep (2026). https://doi.org/10.1038/s41598-026-42977-z
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DOI: https://doi.org/10.1038/s41598-026-42977-z
