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

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

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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.

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

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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.

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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

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  1. Suphansa Sawamiphak
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  2. Mathias Ritter
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  3. Amparo Acker-Palmer
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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.

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Correspondence to Amparo Acker-Palmer.

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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)

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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

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