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

Protocolo

1. Preparing Beads for Gel Implantation

1. Take a culture containing microcarrier beads coated with Human Umbilical Vein Endothelial Cells (HUVEC) and pericytes.
2. 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.
3. 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.
   1. Avoid introducing air bubbles and use a p1000 pipette to minimize shear stress on the endothelial cell-coated beads.
4. Wait 3 min to allow the beads in the solution to settle to the bottom of the tube.
5. Remove as much of the supernatant as possible without disturbing the bead pellet using a p1000 pipette tip (do not use a vacuum aspirator).
6. Add 1 mL of complete media to each tube and resuspend the beads.
7. 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.
8. 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.
9. Add 1 mL of complete media to the beads.

2. Embedding Coated Beads in a Fibrin Gel

1. Remove as much media as possible without disturbing the bead pellet in each FACS tube using a P1000 pipette.
2. Resuspend the beads in 2.5 mL of fibrinogen solution.
3. 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.
4. Add 0.5 mL of bead/fibrinogen solution to each well.
   1. Be sure to resuspend the bead solution well prior to pipetting the solution each time.
   2. 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.
   3. Be sure to change the pipette tip between wells.
   4. Take caution to not move or disturb the plate at any point during initial fibrin clotting, as any movement may disrupt the gel formation.
5. Leave the plate sitting in the hood for 30 min at room temperature.
6. 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

1. During the last 30 min of the gel solidifying, detach Normal Human Lung Fibroblasts (NHLF) as follows:
   1. 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.
   2. 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.
2. Plate 1 mL of the NHLF cell suspension onto the gel of each well and place it back in the incubator.
3. Change complete media every other day for the duration of the assay.

Materiais

Name	Company	Catalog Number	Comments
Sterile Pipette tips	VWR
Pipettors	Eppendorf
Complete EGM2 Media Bullet Kit	Lonza	CC-3162	HUVEC Media
MEM	Gibco	11095114	10T1/2 Media
DMEM	Gibco	11965118	NHLF Media
Tissue culture-grade PBS	Gibco	14190-144	Magnesium and calcium free
Accutase	Life Technologies	A1110501	For lifting HUVEC
Trypsin	Life Technologies	15050065	For lifting 10T 1/2 and NHLF
HUVEC	Lonza	C2517A
10T 1/2	ATCC
NHLF	ATCC
Cytodex 3 microcarrier beads	Sigma	C3275
Tissue culture-coated 6 and 10 cm plates	Corning
Fibrinogen from bovine plasma	Sigma	F8630
Thrombin	Sigma	t9549
Aprotinin	Sigma	a3428
Falcon Round-Bottom Tubes	Corning
Tissue culture incubator and hood
24-well glass bottom plates	MatTek	P24G1.513F	Glass-bottom plates are needed only if the sprouts are going to be imaged. If not, tissue culture plastic is also acceptable.