Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Preparation of retinal explant cultures to study ex vivo tip endothelial cell responses

Nature Protocols volume 5, pages 1659–1665 (2010)Cite this article

Subjects

Abstract

This protocol details a culture technique for neonatal mouse retina that allows the assessment and quantification of acute responses of developing blood vessels to pharmacological manipulation. The technique has proven to be a useful tool for elucidating the molecular mechanisms that underlie the guidance of tip cells in the complex scenario of the angiogenic sprouting process. This culture setting allows the acute stimulation or inhibition of cellular functions of endothelial cells in their physiological environment ex vivo. Compared with other existing techniques, such as retinal injections in animals, the explant culture described here is an easily manageable and highly flexible alternative that allows pharmacological manipulations of the developing retina vessels. The technique involves swift extraction of retina from intact eye and retinal flat mounting on a hydrophilic membrane with minimum disturbance of the tissue. The responses of tip endothelial cell sprouting activity and filopodial extension to different angiogenic and angioinhibitory factors can be evaluated within only 4 h. The whole process for the retinal explant cultures and stimulation can be completed in 10 h.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Retinal dissection and explant culture.
Figure 2: Example of vessel sprout outgrowth assay.

References

  1. Dorrell, M.I. & Friedlander, M. Mechanisms of endothelial cell guidance and vascular patterning in the developing mouse retina. Prog. Retin. Eye Res. 25, 277–295 (2006).

    Article  PubMed  Google Scholar 

  2. Gerhardt, H. et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J. Cell. Biol. 161, 1163–1177 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. le Noble, F., Klein, C., Tintu, A., Pries, A. & Buschmann, I. Neural guidance molecules, tip cells, and mechanical factors in vascular development. Cardiovasc. Res. 78, 232–241 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Sawamiphak, S. et al. EphrinB2 regulates VEGFR2 function in developmental and tumour angiogenesis. Nature 465, 487–491 (2010).

    Article  CAS  PubMed  Google Scholar 

  5. Gariano, R.F. & Gardner, T.W. Retinal angiogenesis in development and disease. Nature 438, 960–966 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Stone, J. et al. Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia. J. Neurosci. 15, 4738–4747 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Carmeliet, P. & Tessier-Lavigne, M. Common mechanisms of nerve and blood vessel wiring. Nature 436, 193–200 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Stalmans, I. et al. Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms. J. Clin. Invest. 109, 327–336 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Fraisl, P., Mazzone, M., Schmidt, T. & Carmeliet, P. Regulation of angiogenesis by oxygen and metabolism. Dev. Cell. 16, 167–179 (2009).

    Article  CAS  PubMed  Google Scholar 

  10. Lu, X. et al. The netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system. Nature 432, 179–186 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Donovan, S.L. & Dyer, M.A. Preparation and square wave electroporation of retinal explant cultures. Nat. Protoc. 1, 2710–2718 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Kretz, A., Marticke, J.K., Happold, C.J., Schmeer, C. & Isenmann, S. A primary culture technique of adult retina for regeneration studies on adult CNS neurons. Nat. Protoc. 2, 131–140 (2007).

    Article  CAS  PubMed  Google Scholar 

  13. Dor, Y., Porat, R. & Keshet, E. Vascular endothelial growth factor and vascular adjustments to perturbations in oxygen homeostasis. Am. J. Physiol. Cell. Physiol. 280, C1367–C1374 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Garin, G. & Berk, B.C. Flow-mediated signaling modulates endothelial cell phenotype. Endothelium 13, 375–384 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Iruela-Arispe, M.L. & Davis, G.E. Cellular and molecular mechanisms of vascular lumen formation. Dev. Cell. 16, 222–231 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank A. Filosa, I. Bethani, K. Happich, D. Schmelzer and U. Bauer for technical support. This work is supported by grants from the Deutsche Forschungsgemeinschaft within SPP1190 (AC180/3-1, 3-2 to A.A-P.), SFB 834 (A7 to A.A.-P.) and the Clusters of Excellence “Macromolecular Complexes (CEF)” (EXC 115) at the University of Frankfurt and “Cardio-Pulmonary System (ECCPS)” (EXC 147) at the Universities of Giessen and Frankfurt.

Author information

Authors and Affiliations

  1. Frankfurt Institute for Molecular Life Sciences (FMLS) and Institute of Cell Biology and Neuroscience, Goethe University Frankfurt, Frankfurt am Main, Germany

    Suphansa Sawamiphak, Mathias Ritter & Amparo Acker-Palmer

Authors
  1. Suphansa Sawamiphak
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mathias Ritter
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Amparo Acker-Palmer
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.S. developed and wrote the protocol and prepared figures. M.R. helped with troubleshooting and assisted S.S. with recording the video. A.A.-P. supervised and interpreted results, prepared figures and wrote the protocol.

Corresponding author

Correspondence to Amparo Acker-Palmer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Retinal explant procedure. The video shows the main steps of the retinal explant procedure including eye enucleation, cornea incision, sclera/choroid/pigment epithelium removal, iris removal, lens/vitreous removal, retina incision, retina transfer and retina flat-mounting. (WMV 17704 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sawamiphak, S., Ritter, M. & Acker-Palmer, A. Preparation of retinal explant cultures to study ex vivo tip endothelial cell responses. Nat Protoc 5, 1659–1665 (2010). https://doi.org/10.1038/nprot.2010.130

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.130

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing