In Vitro Spheroid Sprouting Assay of Angiogenesis

Methods Mol Biol. 2019:1952:211-218. doi: 10.1007/978-1-4939-9133-4_17.


Angiogenesis is a well-coordinated physiological process that leads to new blood vessel formation. Physiologically, angiogenesis is more prominent during development and wound healing and its dysregulation drives or is related to several diseases, including cancer. The endothelial cells are the main regulators of the angiogenic process, and thus the angiogenic outcome is assessed based on the effect on endothelial cell functions. Several in vitro and in vivo techniques have been developed to assess the effect of various factors on angiogenesis. Compared to the in vivo techniques, the in vitro techniques are considered less physiologically relevant. This has been partially overcome by the development of 3-dimensional (3D) in vitro models, one of which is the spheroid assay or 3D sprouting assay that exploits the effect of the extracellular matrix to endothelial cell functions. This chapter focuses on the description of the spheroid assay and mentions the variations and potential applications this assay can have.

Keywords: Angiogenesis; Collagen; Endothelial cells; Spheroids; Sprouts.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Culture Techniques / methods*
  • Collagen / metabolism
  • Endothelial Cells / cytology
  • Endothelial Cells / physiology*
  • Endothelial Cells / ultrastructure
  • Extracellular Matrix / metabolism*
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Microscopy, Confocal / methods
  • Neovascularization, Physiologic*
  • Spheroids, Cellular / cytology
  • Spheroids, Cellular / physiology*
  • Spheroids, Cellular / ultrastructure
  • Staining and Labeling / methods


  • Collagen