To begin, add HUVECs into a 10 milliliter Falcon tube containing eight milliliters of endothelial growth medium. Add two milliliters of METHOCEL stock solution into the tube and shake well. Next, transfer 25 microliters of the mixture onto the inverted inner surface of a large, square Petri dish.
Gently rotate the lid by 180 degrees and place it on the bottom of a cell culture vessel and incubate overnight. The next day, add 2.3 milliliters of rat tail collagen Type I to a vial containing 0.28 milliliters of 10X Medium 199. Keep this mixture on ice and mix until it is evenly yellow.
Titrate the mixture against sodium hydroxide until it turns orange. Then, add 50 microliters of HEPES buffer to the final product. Collect the hanging cultured cells into a 50 milliliter Falcon tube with 20 milliliters of PBS, then centrifuge.
After discarding the supernatant, add 0.1 milliliters of FBS and 0.4 milliliters of endothelial basal medium to the spheroid pellet. Gently tap the tube to resuspend the pellet. Now, pipette two milliliters of METHOCEL stock solution and mix well.
Then, add two milliliters of the prepared collagen mixture to the resuspended spheroids. Dispense 0.5 milliliters of the resulting mixture into the wells of a 24-well plate and incubate. Next, pipette 100 microliters of diluted recombinant human vascular endothelial growth factor into the wells of the plate.
Incubate the cell culture at 37 degrees Celsius under 5%carbon dioxide supplementation for 12 hours. The next day, with an inverted microscope, capture the images of the spheroids that are not in contact with the well rim, with each other, or show signs of damage. The spheroids stimulated with only basal medium or recombinant human vascular endothelial growth factor were clearly distinguishable.
Spheroids in low-quality gels appeared to drop to the ground and dispersed. The negative control showed a moderate baseline sprouting rate while the human vascular endothelial growth factor doubled or tripled the relative sprouting length. The number of sprouts showed a similar dynamic range.
