To begin, obtain 250 milligrams of SAT above the pectorals major muscle during CIED implantation in magnetic-activated cell-sorting tissue storage solution. Prepare one milligram per milliliter of Collagenase/Dispase in 10 milliliters of cold Hanks'Balanced Salt Solution. Under a laminar flow cabinet, mince tissues into one-cubic millimeter pieces in 500 microliters of Collagenase/Dispase solution.
Add 4.5 milliliters of the Collagenase/Dispase solution to the mix and transfer the triturated tissue to a clean 50-milliliter centrifuge tube. Wash the Petri dish lid with five milliliters of Collagenase/Dispase solution and add it to the tube. Incubate the tube for 30 minutes at 37 degrees Celsius in a tube rotator set to 20 revolutions per minute.
To halt the digestion, pour 10 milliliters of complete endothelial cell growth media into the tube. After triturating the digested mix with a 14-gauge Venflon cannula, strain it through a 70-micrometer cell sieve and rinse it with 10 milliliters of 0.5%PBS-BSA buffer. Centrifuge the filtrate at 300G for 10 minutes at room temperature and discard the supernatant.
To remove the dead cells, add 200 microliters of dead cell removal beads to the cell pellet taken in a microcentrifuge tube, and incubate. Then attach a liquid-separation column with a 30-micrometer filter to the separator magnet and place a 15-milliliter centrifuge tube underneath. After washing the column with dead cell removal binding buffer, add the sample bead suspension.
Let it pass through under gravity and collect the eluate. Next, spin the collected live cell eluate and resuspend the resulting pellet in 400 microliters of 0.5%PBS-BSA. Incubate the suspended cells with 20 microliters of anti-CD31-coated magnetic beads at four degrees Celsius for 20 minutes.
After centrifuging, resuspend the cell pellet in 500 microliters of 0.5%PBS-BSA. Attach a column with a 30-micrometer filter to the separator magnet and place a 15-milliliter centrifuge tube underneath. Add the cell microbead suspension to the column and let it pass through under gravity.
After washing the column, place it in a fresh 15-milliliter centrifuge tube and add one milliliter of 0.5%PBS-BSA to the column. Push the column plunger to collect the CD31-positive fraction. Centrifuge the sample as demonstrated earlier and resuspend the cell pellet in one milliliter of complete microvascular endothelial cell growth media.
Dispense the suspension into two wells of a 2%gelatin-coated 24-well plate. Culture the cells at 37 degrees Celsius with 5%carbon dioxide for two to four weeks until cells become nearly 80%confluent.
