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.

Examine the dishes of microcarrier-coated beads under the microscope at 20 times magnification to confirm that all of the beads have been sufficiently coated by the endothelial cells.

Transfer the plates to a laminar flow hood, and use a P1000 pipette to vigorously aspirate and dispense the coated-beads solutions to detach the cells from the plate bottoms, and transfer the supernatants into individual tubes.

Allow the beads to settle to the bottoms of the tubes for three minutes. Then, use the P1000 pipette to remove as much of the supernatants as possible without disturbing the bead clusters. Resuspend the beads in one milliliter of complete medium per tube, and allow the beads to settle for 30 to 60 seconds. Use the P1000 pipette to aspirate the supernatant again, and wash the free-floating endothelial cells from the bead suspension with fresh complete medium, two more times, as just demonstrated.

After the third wash, resuspend the beads in 2.5 milliliters of fibrinogen solution. Add 13 microliters of thrombin solution per well, in a 24-well glass bottom plate. Then, add 0.5 milliliters of the bead/fibrinogen solution to each well, taking care to carefully pipette the beads directly into the thrombin, and slowly pipette up and down two to three times to thoroughly mix the solutions.

When all of the wells have been seeded, allow the thrombin-fibrinogen solution to pre-clot in the laminar flow hood for 30 minutes at room temperature. At the end of the incubation, carefully transfer the plate to a 37-degree Celsius incubator for an additional 1.5 to 2 hours.

When the gel has fully solidified, resuspend normal human lung fibroblasts at 2 x 10⁴ cells per milliliter concentration in complete medium, and add one milliliter of fibroblasts to each well. Then, return the plate to the cell culture incubator.