Animal-origin-free method for generating blood vessel organoids

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Animal-origin-free method for generating blood vessel organoids

Scientific Reports , Article number:  (2026) Cite this article

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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.

Data availability

The data, presented in this study, are available from the corresponding author on reasonable request.

References

  1. Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459(7244), 262–5 (2009).

    Google Scholar 

  2. Huch, M. et al. In vitro expansion of single Lgr5 + liver stem cells induced by Wnt-driven regeneration. Nature 494(7436), 247–50 (2013).

    Google Scholar 

  3. Sachs, N. et al. Long-term expanding human airway organoids for disease modeling. EMBO J. https://doi.org/10.15252/embj.2018100300 (2019).

    Google Scholar 

  4. Boj, S. F. et al. Organoid models of human and mouse ductal pancreatic cancer. Cell 160 (1–2), 324–338 (2015).

    Google Scholar 

  5. Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501(7467), 373–9 (2013).

    Google Scholar 

  6. Wimmer, R. A., Leopoldi, A., Aichinger, M., Kerjaschki, D. & Penninger, J. M. Generation of blood vessel organoids from human pluripotent stem cells. Nat. Protoc. 14(11), 3082–100 (2019).

    Google Scholar 

  7. Wimmer, R. A. et al. Human blood vessel organoids as a model of diabetic vasculopathy. Nature 565(7740), 505–10 (2019).

    Google Scholar 

  8. Kong, D. et al. In vitro modeling of atherosclerosis using iPSC-derived blood vessel organoids. Adv. Healthc. Mater. 14(1), e2400919 (2025).

    Google Scholar 

  9. Aisenbrey, E. A. & Murphy, W. L. Synthetic alternatives to Matrigel. Nat. Rev. Mater. 5(7), 539–51 (2020).

    Google Scholar 

  10. Li, K., He, Y., Jin, X., Jin, K. & Qian, J. Reproducible extracellular matrices for tumor organoid culture: Challenges and opportunities. J. Transl. Med. 23(1), 497. (2025).

    Google Scholar 

  11. Kozlowski, M. T., Crook, C. J. & Ku, H. T. Towards organoid culture without Matrigel. Commun. Biol. 4(1), 1387. (2021).

    Google Scholar 

  12. Brassard, J. A. & Lutolf, M. P. Engineering stem cell self-organization to build better organoids. Cell Stem Cell 24(6), 860–76 (2019).

    Google Scholar 

  13. Schutgens, F. & Clevers, H. Human organoids: Tools for understanding biology and treating diseases. Annu. Rev. Pathol. 15, 211–234 (2020).

    Google Scholar 

  14. Soto-Gamez, A., Gunawan, J. P., Barazzuol, L., Pringle, S. & Coppes, R. P. Organoid-based personalized medicine: From tumor outcome prediction to autologous transplantation. Stem Cells 42(6), 499–508 (2024).

    Google Scholar 

  15. Naderi-Meshkin, H. et al. Unveiling impaired vascular function and cellular heterogeneity in diabetic donor-derived vascular organoids. Stem Cells 42(9), 791–808 (2024).

    Google Scholar 

  16. Ahn, Y. et al. Blood vessel organoids generated by base editing and harboring single nucleotide variation in Notch3 effectively recapitulate CADASIL-related pathogenesis. Mol. Neurobiol. 61(11), 9171–83 (2024).

    Google Scholar 

  17. Gjorevski, N. et al. Designer matrices for intestinal stem cell and organoid culture. Nature 539(7630), 560–4 (2016).

    Google Scholar 

  18. Giles, R. et al. Animal-free alternatives for Matrigel in human iPSC-derived blood vessel organoid culture. Sci. Rep. 15(1), 36042 (2025).

    Google Scholar 

  19. Brandenberg, N. et al. High-throughput automated organoid culture via stem-cell aggregation in microcavity arrays. Nat. Biomed. Eng. 4 (9), 863–874 (2020).

    Google Scholar 

  20. Quintard, C. et al. A microfluidic platform integrating functional vascularized organoids-on-chip. Nat. Commun. 15 (1), 1452 (2024).

    Google Scholar 

  21. Tubbs, E. et al. Human assembloid of human blood vessel organoids with pancreatic islets improves insulin secretion over time ex vivo. Cell Rep. 44(10), 116378 (2025).

    Google Scholar 

  22. Heo, J. H., Kang, D., Seo, S. J. & Jin, Y. Engineering the extracellular matrix for organoid culture. Int. J. Stem Cells 15(1), 60–9 (2022).

    Google Scholar 

  23. Moss, S. P., Bakirci, E. & Feinberg, A. W. Engineering the 3D structure of organoids. Stem Cell Reports 20(1), 102379 (2025).

    Google Scholar 

  24. Nwokoye, P. N. & Abilez, O. J. Bioengineering methods for vascularizing organoids. Cell Rep. Methods 4(6), 100779 (2024).

    Google Scholar 

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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.

Author information

Authors and Affiliations

  1. Angios FlexCo, Exlgasse 24, 6020, Innsbruck, Austria

    Alexander Hoffmann, David Schorn, Jakob Thönig, Yu-Hsiang Teng & Teodor E. Yordanov

  2. Department of Internal Medicine II, Medical University Innsbruck, Innsbruck, Austria

    Alexander Hoffmann

  3. Department of Dermatology, ETAP-Lab, Vandoeuvre-lès-Nancy, France

    Jean-François Bisson

Authors

  1. Alexander Hoffmann
  2. David Schorn
  3. Jakob Thönig
  4. Yu-Hsiang Teng
  5. Jean-François Bisson
  6. Teodor E. Yordanov

Contributions

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.

Corresponding author

Correspondence to Teodor E. Yordanov.

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The authors declare no competing interests.

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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

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