Human Endothelial Cell Spheroid-based Sprouting Angiogenesis Assay in Collagen

Commonly used acronym: In vitro sprouting assay

Scope of the method

The Method relates to
  • Human health
The Method is situated in
  • Basic Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Human derived cells / tissues / organs
Specify the type of cells/tissues/organs
HUVEC (Human Umbilical Vein Endothelial Cell) or HDLEC (Human Dermal Lymphatic Endothelial Cell)


Method keywords
  • (lymph)angiogenesis
  • Endothelial cells
  • growth factor supplementation
  • spheroids
Scientific area keywords
  • vascular development
  • blood vessel
  • tumor stroma
  • sprouting angiogenesis
Method description

This assay evaluates the sprouting ability of endothelial cells in a collagen matrix and it used as an in vitro model to study the formation of new blood/lymphatic vessels. 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. 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,
  • - Microscope.
Method status
  • History of use
  • Internally validated
  • Published in peer reviewed journal

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.

Future & Other applications

This method could also be used using conditioned media from other cell types (for example cancer cells). As such, it could be used to asses the amount of (lymph)angiogenic signaling in the secreted medium.

References, associated documents and other information

  • Meçe, O., Houbaert, D., Sassano, ML. et al. Lipid droplet degradation by autophagy connects mitochondria metabolism to Prox1-driven expression of lymphatic genes and lymphangiogenesis. Nat Commun 13, 2760 (2022).

Contact person

Diede Houbaert


Cellular and Molecular Medicine - KU Leuven
Department of Cellular and Molecular Medicine
Laboratory of Cell Death Research & Therapy (VIB-KU Leuven)