An optimized three-dimensional in vitro model for the analysis of angiogenesis.

Angiogenesis is the formation of new blood vessels from the existing vasculature. It is a multistage process in which activated endothelial cells (EC) degrade basement membrane, sprout from the parent vessel, migrate, proliferate, align, undergo tube formation, and eventually branch and anastomose with adjacent vessels. Here we describe a three-dimensional in vitro assay that reproduces each of these steps. Human umbilical vein endothelial cells (HUVEC) are cultured on microcarrier beads, which are then embedded in a fibrin gel. Fibroblasts cultured on top of the gel provide factors that synergize with bFGF and VEGF to promote optimal sprouting and tube formation. Sprouts appear around day 2, lumen formation begins at day 4, and at day 10 an extensive anastomosing network of capillary-like tubes is established. The EC express a similar complement of genes as angiogenic EC in vivo and undergo identical morphologic changes during tube formation. This model, therefore, recapitulates in vivo angiogenesis in several critical aspects and provides a system that is easy to manipulate genetically, can be visualized in real time, and allows for easy purification of angiogenic EC for downstream analysis.