1. Preparing Beads for Gel Implantation
- Take a culture containing microcarrier beads coated with Human Umbilical Vein Endothelial Cells (HUVEC) and pericytes.
- Examine the dishes containing the microcarrier-coated beads under the microscope using the 20X objective to ensure that all beads have been sufficiently coated by endothelial cells.
- Vigorously pipette the plated solution of cells to detach them from the 6-cm plate and place the solution into a tube. Wash the plate 2X additional times with complete media and transfer to the tube to collect all residual beads that may be adhered to the plate.
- Avoid introducing air bubbles and use a p1000 pipette to minimize shear stress on the endothelial cell-coated beads.
- Wait 3 min to allow the beads in the solution to settle to the bottom of the tube.
- Remove as much of the supernatant as possible without disturbing the bead pellet using a p1000 pipette tip (do not use a vacuum aspirator).
- Add 1 mL of complete media to each tube and resuspend the beads.
- Wait 30-60 s to allow the beads to settle to the bottom. Then remove as much of the supernatant as possible using a P1000 pipette without disturbing the bead pellet. Do not use a vacuum aspirator as the likelihood of accidentally removing the bead pellet is greatly increased.
- Repeat Steps 1.6 and 1.7 two additional times. The goal is to remove as much of the free-floating HUVEC that may have been detached from the 6-cm plate as possible, while not removing too many HUVEC-coated beads, which should settle in the round-bottomed Fluorescence Activated Cell Sorting (FACS) tubes more quickly than free-floating cells.
- Add 1 mL of complete media to the beads.
2. Embedding Coated Beads in a Fibrin Gel
- Remove as much media as possible without disturbing the bead pellet in each FACS tube using a P1000 pipette.
- Resuspend the beads in 2.5 mL of fibrinogen solution.
- Pipette 13 µL of thrombin solution into each desired well of a 24-well glass-bottomed plate. Plating in a glass-bottomed plate is essential for enabling optimal imaging of the sprouts.
NOTE: Do not leave thrombin sitting in a well for more than 5-10 min before the next step.
- Add 0.5 mL of bead/fibrinogen solution to each well.
- Be sure to resuspend the bead solution well prior to pipetting the solution each time.
- Take caution to carefully pipette directly into the thrombin already present in the well. Slowly pipette up and down 2-3 times to thoroughly mix the solution, taking caution to not introduce any air bubbles.
- Be sure to change the pipette tip between wells.
- Take caution to not move or disturb the plate at any point during initial fibrin clotting, as any movement may disrupt the gel formation.
- Leave the plate sitting in the hood for 30 min at room temperature.
- Carefully transfer the plate to a 37 °C incubator for an additional 1.5-2 h to allow the gel to fully solidify
3. Plating Fibroblasts on Top of the Fibrin Gel
- During the last 30 min of the gel solidifying, detach Normal Human Lung Fibroblasts (NHLF) as follows:
- Wash a T175 flask of cells with 10 mL of Phosphate Buffered Saline (PBS), then add 5 mL of cell detachment solution and incubate at 37 °C for 10 min.
- Resuspend NHLF at a concentration of 20,000 cells/mL in complete media.
NOTE: NHLF cells can be cultured with Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin Streptomycin.
- Plate 1 mL of the NHLF cell suspension onto the gel of each well and place it back in the incubator.
- Change complete media every other day for the duration of the assay.
