Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

The overall goal of this assay is to incorporate heterotypic cellular interactions in vitro that encapsulate the complexity of angiogenesis in vivo. This method can help answer key questions in the vascular biology field about the importance of pericyte endothelial cell interactions during angiogenesis. The main advantage of this technique is that it integrates the different stages of sprouting angiogenesis, including proliferation, migration and anastomosis while still permitting the study of endothelial cell pericyte interactions.

Begin by reconstituting the microcarrier beads at a six times 10 to the fourth beads per milliliter concentration in PBS, followed by autoclave sterilization of the microcarrier bead suspension. Next, aliquot one milliliter of complete medium into the appropriate number of round-bottom Fluorescence Activated Cell Sorting, or FACS, tubes, and add 20 microliters of room temperature-cooled, autoclave sterilized microcarrier bead suspension to each tube. Add one milliliter of cells to each tube.

Agitate tubes and incubate the tubes at 37 degrees Celsius for 2 1/2 to three hours with gentle agitation every 20 minutes. At the end of the incubation, rest the cells in beads at room temperature for five minutes to allow the cells and beads to settle to the bottom of the tubes. Then, use a P1000 pipette to replace 0.5 milliliters of medium from each tube with two times 10 to the fifth pericytes and 0.5 milliliters of complete medium.

Agitate the cell-bead suspension, and return the cells to the 37 degrees Celsius incubator with gentle agitation every 20 minutes until the beads have been agitated for a total of four hours. At the end of the incubation, gently agitate the tubes one last time, and immediately transfer the entire two milliliter volume of solution from each tube into individual six centimeter plates. Then, add an additional three milliliters of complete medium to each plate, and incubate the plates at 37 degrees Celsius overnight.

The next morning, examine the dishes of microcarrier-coated beads under the microscope at a 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-bead 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. Re-suspend 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, re-suspend 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 preclot 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 two hours. When the gel has fully solidified, re-suspend normal, human lung fibroblasts at a two times 10 to the fourth 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. Strict adherence to the coating protocol will result in cell-coated beads with a rough, golf ball-like appearance, as illustrated in this representative image of sufficiently coated beads that are ready for fibrin gel implantation. Further, this protocol facilitates a tight association of the two cell types in vitro with the presence of the pericytes complementing the occurrence of sprouting.

While attempting this procedure, it is important to remember to completely coat the beads with cells during the agitation steps, and to carefully embed the beads in the fibrin gel without introducing bubbles during the gel implantation. Following this procedure, other analyses like mouse studies of angiogenesis can be performed to answer additional questions, such as what is the in vivo relevance of the mechanisms and phenotypes explored in the pericyte beads sprouting assay in vitro? This technique will be useful for researchers in the field of cancer biology and exploring endothelial cell pericyte interactions and functions in a more physiologically relevant in vitro model of angiogenesis.