In vitro endothelial spheroid sprouting assay for angiogenesis

Scope of the method

The Method relates to
  • Animal health
  • Human health
The Method is situated in
  • Basic Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Animal derived cells / tissues / organs
Species from which cells/tissues/organs are derived
Type of cells/tissues/organs


Method keywords
  • collagen
  • endothelial
  • sprouting
  • spheroids
Scientific area keywords
  • angiogenesis
  • tip cell
  • endothelial sprouts
  • sprouting angiogenesis
Method description

This method evaluates sprouting propensity of endothelial cells in a collagen matrix. The effect of pro-angiogenic growth factors or co-cultured cells can be measured by quantifying the amount of vascular sprouts that form on endothelial spheroids. Endothelial spheroids are obtained by growing endothelial cells in hanging drops, which forces the cells to adhere to each other. The peripheral cells acquire a flat phenotype, similar to the flattened endothelial wall of blood vessels. After generation of the spheroids, they are embedded in a collagen matrix in which endothelial growth factors or specific cell types can be embedded. Finally the amount of endothelial sprouts is quantified as a measure of the endothelial sprouting propensity.

Lab equipment
  • Biosafety cabinet ;
  • CO2 incubator ;
  • Inverted microscope.
Method status
  • Internally validated

Pros, cons & Future potential


The method is simple and the vascular sprouts share multiple morphological characteristics of vascular tip cells in vivo.


The model is limited to evaluating sprout propensity, which is only the first step in the angiogenic cascade. The subsequent steps of tubule and network formation cannot be evaluated.


Spheroids can also be made from a mix of cells. We have performed this with hepatic cancer cells and this lead to a core of cancer cells surrounded by a flattened endothelial layer, similar to an inside out blood vessel.

References, associated documents and other information


Ffisterer, L. Korff, T. (2016) Spheroid-Based In Vitro Angiogenesis model. Methods Mol Biol; 1430:167-77 doi: 10.1007/978-1-4939-3628-1_11

An alternative spheroid sprouting protocol

Contact person

Ward De Spiegelaere


Ghent University (UGent)
Flemish Region