Bead Sprouting Assay for Early-Stage Angiogenesis Assessment: An In Vitro Bead-Based Model to Study Endothelial Sprouting in the Presence of Pericytes

Angiogenesis is the formation of new blood vessels from the existing blood vessels during developmental and pathological conditions. Extensive communication between the endothelial cells that line the inner surface of blood vessels and the surrounding pericytes - supporting cells on the outer side of blood vessels - is crucial for angiogenesis.

To study in vitro angiogenesis, begin with endothelial cells and pericytes-coated microcarrier beads suspended in a fibrinogen solution - a soluble glycoprotein that acts as a fibrin precursor.

Now, pipette the bead suspension into a culture plate containing a solution of thrombin - a proteolytic enzyme, and incubate. Thrombin binds to the fibrinogen molecule, causing its cleavage to form insoluble fibrin monomers, forming polymeric fibrin gel. This step ensures the embedding of cell-coated beads in the fibrin gel.

Next, plate a culture of fibroblasts - connective tissue precursor cells - on top of the fibrin gel and incubate to allow fibroblasts' activation.

During incubation, the fibroblasts secrete crucial angiogenic-growth factors that trigger endothelial cell activation. In combination, adherent pericytes and endothelial cells secrete signaling molecules that help in endothelial cell sprouting in the fibrin gel.

The endothelial sprouts appear as pericyte-wrapped elongated cell protrusions, depicting the early stage of angiogenesis.