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In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract

Abstract

A protocol is presented here for a rapid, quantitative and reliable in vitro angiogenesis assay that can be adapted for high throughput use. Endothelial cells are plated on a gelled basement matrix, their natural substrate, and form capillary-like structures with a lumen. The assay can be used to identify inhibitors or stimulators of angiogenesis, as well as genes and signaling pathways involved in angiogenesis. It has also been used to identify endothelial progenitor cells. This assay involves endothelial cell adhesion, migration, protease activity and tubule formation. This tube formation assay is preferred, as other in vitro assays for angiogenesis, such as cell adhesion, migration and invasion, measure limited steps in the angiogenesis process. The tube formation assay on basement membrane can be completed in a day because transformed endothelial cells form tubes within 3 h, whereas non-transformed endothelial cells form tubes within 6 h.

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Figure 1: Dynamics of the behavior of endothelial cells (HUVECs) after plating on a basement membrane substratum.
Figure 2: Effect of angiogenesis stimulators and an inhibitor on tube formation.

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Authors and Affiliations

Authors

Contributions

I.A. designed and performed the experiments and wrote the paper. H.K.K. designed the experiments and wrote the paper.

Corresponding authors

Correspondence to Irina Arnaoutova or Hynda K Kleinman.

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Competing interests

I.A. is an employee of Trevigen Inc., and H.K. is a consultant for Trevigen Inc. which is one of the companies commercializing the basement membrane extract (Matrigel, Cultrex).

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Arnaoutova, I., Kleinman, H. In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nat Protoc 5, 628–635 (2010). https://doi.org/10.1038/nprot.2010.6

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