The protocol demonstrates a reliable and reproducible method to isolate and culture high purity endothelial cells from mice that could have multiple applications. This technique is a more straightforward method that preserves the yield and purity of endothelial cells. Demonstrating the procedure will be Nina Nguyen, a staff research associate from my laboratory.
To begin, vortex the magnetic beads for a few seconds. Pipette 50 microliters of the beads into two 1.5 milliliter microcentrifuge tubes for CD31 and CD102. Add one milliliter of bead wash buffer and mix well.
Place the tubes on a magnetic separator and remove the supernatant with a glass Pasteur pipette. Resuspend the beads in 50 microliters of bead wash buffer. Then add five microliters or 2.5 micrograms of CD31 or CD102 antibody to every 50 microliters of beads.
Incubate bead suspension with gentle rotation on an end over end rotator overnight at four degrees Celsius or for three hours at room temperature. Wash immunobeads four times and then resuspend the immunobeads in 50 microliters of bead wash buffer. On the day of isolation, add 25 milliliters of HBSS to 25 milligrams of type one collagenase and incubate it with gentle rotation.
Then filter using a 22 micrometer filter. After euthanizing a five to seven-day-old mouse pup, spray the carcass with 70%ethanol. Next, cut the diaphragm superiorly upwards along the entire lateral walls of the chest to expose the thoracic cavity.
Inject five milliliters of cold DMEM into the heart's right ventricle until the lungs turn white. Then cut the lobes distal to the corresponding bronchi one by one to remove the lungs. Pull the lung lobes in a 50 milliliter conical tube prefilled with 20 milliliters of ice cold basal medium.
Gently agitate the tube by hand for 10 to 15 seconds to wash any excess red blood cells. Remove the lungs from the media with a cell strainer and mince with sterilized scissors. Transfer the minced tissue to the 50 milliliter conical tube with 25 milliliters of prewarmed collagenase solution.
Gently agitate on a rotating mixer. Attach a 20 milliliter syringe to a 15 gauge blunt cannula and triturate the suspension vigorously without frothing 10 to 15 times into a cellular suspension. Filter the suspension through a 70 micrometer cell strainer into a 50 milliliter conical tube.
Then rinse the conical tube used for the digestion and the cell strainer with 15 milliliters of basal medium. Centrifuge the cellular suspension at 400 times G for eight minutes at four degrees Celsius. Resuspend the pellet in two milliliters of complete medium.
Add eight milliliters of complete medium and incubate it. The following day, wash flasks twice with 10 milliliters of HBSS without calcium and magnesium and add 10 milliliters of complete medium. Once cells are 90 to 95%confluent, they are ready for primary immunobead isolation.
Wash adherent endothelial cells twice with 10 milliliters of HBSS without calcium and magnesium. Add two milliliters of trypsin-free cell detachment solution and incubate it to ensure the lifting of all the cells from the flask. Add eight milliliters of basal medium.
Next, spin down the cells at 400 times G for four minutes at room temperature and resuspend them in two milliliters of the basal medium. Vortex anti-CD31 coated beads for a few seconds to resuspend the beads. Add 30 microliters of beads for every two milliliters of cell suspension and secure the lid.
Incubate tube for 10 minutes at room temperature on an end over end rotator. Then place the tubes on a magnetic separator for two minutes. Aspirate the supernatant and remove the tube from the magnet.
Resuspend the bead cell pellet by adding three milliliters of basal medium to the tube and pipetting up and down several times. Replace the tube on the magnetic separator for two minutes. Then carefully aspirate the supernatant using a glass Pasteur pipette.
After the final wash, when the supernatant becomes clear, resuspend the bead cell pellet in three milliliters of complete growth medium and transfer it to a T75 flask. Add seven milliliters of the complete growth medium and incubate it. On the next day, rewash the cells with HBSS without calcium and magnesium.
Then add 10 milliliters of complete medium. Once cells are 90 to 95%confluent, they are ready for secondary immunobead isolation. The first immunobead selection identifies the CD31 positive cells, which grow in a cobblestone formation.
A second immunobead selection with anti-CD102 coated beads increases endothelial cell purity and fluorescence activated cell sorting is used to confirm that the cell population is CD31 positive and CD102 positive. To study endothelial inflammatory pathways and endothelial barrier function, treatment with murine Cytomix was used to induce significant IL-6 production by endothelial cells with a P value of less than 0.01. Moreover, transendothelial resistance decreased showing increased endothelial permeability.
Isolated endothelial cells can be used in endothelial cell stimulations or barrier function assays to discover endothelial-based therapeutic targets for endothelial dysfunction disorders.
