Spheroid-based human endothelial cell microvessel formation in vivo (original) (raw)

Nature Protocols volume 4, pages 1202–1215 (2009)Cite this article

Abstract

The study of angiogenic endothelial cells (ECs) has in recent years greatly stimulated multiple fields of vascular biology research. A number of cellular models and numerous complex developmental, manipulatory and tumor animal models have been developed to study angiogenesis in vitro and in vivo. To connect the versatility of cellular assays with the complexity of readouts of in vivo experimentation, we have developed an endothelial transplantation assay. This assay is based on grafting ex vivo generated EC spheroids (2 d) in a suitable matrix in immunocompromised mice, to give rise to a 3D network of capillaries (20 d). This vasculature connects to the mouse vasculature, is perfused and matures by recruiting mouse mural cells. Here we describe the detailed protocol for this assay, including generation of spheroids, injection into mice, excision and processing of resulting plugs, and quantification by immunohistochemical analysis of the resulting vasculature.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 12 print issues and online access

$259.00 per year

only $21.58 per issue

Buy this article

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

References

  1. Adams, R.H. & Alitalo, K. Molecular regulation of angiogenesis and lymphangiogenesis. Nat. Rev. Mol. Cell Biol. 8, 464–478 (2007).
    Article CAS Google Scholar
  2. Risau, W. Mechanisms of angiogenesis. Nature 386, 671–674 (1997).
    Article CAS Google Scholar
  3. Kerbel, R.S. Tumor angiogenesis. N. Engl. J. Med. 358, 2039–2049 (2008).
    Article CAS Google Scholar
  4. Eichmann, A., Makinen, T. & Alitalo, K. Neural guidance molecules regulate vascular remodeling and vessel navigation. Genes Dev. 19, 1013–1021 (2005).
    Article CAS Google Scholar
  5. Neufeld, G. & Kessler, O. The semaphorins: versatile regulators of tumour progression and tumour angiogenesis. Nat. Rev. Cancer 8, 632–645 (2008).
    Article CAS Google Scholar
  6. Gerhardt, H. & Betsholtz, C. Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res. 314, 15–23 (2003).
    Article Google Scholar
  7. Ellis, L.M. & Hicklin, D.J. VEGF-targeted therapy: mechanisms of anti-tumour activity. Nat. Rev. Cancer 8, 579–591 (2008).
    Article CAS Google Scholar
  8. Carmeliet, P. Angiogenesis in life, disease and medicine. Nature 438, 932–936 (2005).
    Article CAS Google Scholar
  9. Augustin, H.G. Methods in Endothelial Cell Biology (Springer, Berlin, 2004).
  10. Ausprunk, D.H., Knighton, D.R. & Folkman, J. Vascularization of normal and neoplastic tissues grafted to the chick chorioallantois. Role of host and preexisting graft blood vessels. Am. J. Pathol. 79, 597–618 (1975).
    CAS PubMed PubMed Central Google Scholar
  11. Korff, T. & Augustin, H.G. Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation. J. Cell Biol. 143, 1341–1352 (1998).
    Article CAS Google Scholar
  12. Norrby, K. In vivo models of angiogenesis. J. Cell Mol. Med. 10, 588–612 (2006).
    Article CAS Google Scholar
  13. Passaniti, A. et al. A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor. Lab. Invest. 67, 519–528 (1992).
    CAS PubMed Google Scholar
  14. Jain, R.K., Schlenger, K., Hockel, M. & Yuan, F. Quantitative angiogenesis assays: progress and problems. Nat. Med. 3, 1203–1208 (1997).
    Article CAS Google Scholar
  15. Alajati, A. et al. Spheroid-based engineering of a human vasculature in mice. Nat. Methods 5, 439–445 (2008).
    Article CAS Google Scholar
  16. Wong, C., Inman, E., Spaethe, R. & Helgerson, S. Fibrin-based biomaterials to deliver human growth factors. Thromb. Haemost. 89, 573–582 (2003).
    Article CAS Google Scholar
  17. Coughlin, S.R. Thrombin signalling and protease-activated receptors. Nature 407, 258–264 (2000).
    Article CAS Google Scholar
  18. Kleinman, H.K. Preparation of gelled substrates. Curr. Protoc. Cell Biol. Chapter 10, Unit 10.3 (2001).

Download references

Acknowledgements

This work was supported by grants from the German Research Council (DFG, AU83/10-1) and the European Union [LSHG-CT-2004-503573]. HGA is supported by an endowed chair from the Aventis Foundation. Holger Weber is an employee of ProQinase GmbH.

Author information

Author notes

  1. Anna M Laib and Arne Bartol: These authors contributed equally to this study.

Authors and Affiliations

  1. Joint Research Division Vascular Biology of the Medical Faculty Mannheim (CBTM), University of Heidelberg, and the German Cancer Research Center Heidelberg (DKFZ), Im Neuenheimer Feld, Heidelberg, Germany
    Anna M Laib, Arne Bartol & Hellmut G Augustin
  2. Friedrich-Miescher-Institute for Biomedical Research, Maulbeerstrasse, Basel, Switzerland
    Abdullah Alajati
  3. Department of Physiology and Pathophysiology, University of Heidelberg, Im Neuenheimer Feld, Heidelberg, Germany
    Thomas Korff
  4. ProQinase GmbH, Breisacher Straße, Freiburg, Germany
    Holger Weber

Authors

  1. Anna M Laib
  2. Arne Bartol
  3. Abdullah Alajati
  4. Thomas Korff
  5. Holger Weber
  6. Hellmut G Augustin

Corresponding author

Correspondence toHellmut G Augustin.

Rights and permissions

About this article

Cite this article

Laib, A., Bartol, A., Alajati, A. et al. Spheroid-based human endothelial cell microvessel formation in vivo.Nat Protoc 4, 1202–1215 (2009). https://doi.org/10.1038/nprot.2009.96

Download citation

This article is cited by