In Vitro Co-culture of Fibroblast and Endothelial Cells to Assess Angiogenesis.

Abstract

Angiogenesis relies on the spatial and temporal coordination of endothelial migration and proliferation to form new blood vessels. This occurs through synchronous activation of multiple downstream pathways which facilitate vascular development. Proangiogenic growth factors and supporting extracellular matrix allow the formation of capillary-like tubules, reminiscent of microvascular beds, in vitro. In this chapter, we describe a methodology for the establishment of vascular networks by co-culture of endothelial cells and fibroblasts to facilitate the study of tubulogenic and angiogenic potential. We detail the use of siRNA mediated knockdown to deplete target genes of interest, in either the endothelial or fibroblast cells, to allow the assessment of their role in angiogenesis. Finally, we detail how these vascular networks may be stained using immunofluorescence to allow quantification of angiogenic potential in vitro.