Porcine Aortic Endothelial Cell (pAEC) Isolation: An Enzyme-based Method to Isolate Endothelial Cells from Harvested Porcine Abdominal Aorta
Transkript
For porcine aortic endothelial cell isolation, transfer the pig aorta into 150-millimeter Petri dish under aseptic conditions, and gently remove both ends of the vessel. Trim any additional excess tissue where the vessel had been clamped, with sterile forceps and scissors, and use 20 to 50 milliliters of fresh washing buffer to rinse the outside and inside of the aorta.
Next, pass a surgical suture through the aorta, and loosely tie one end of the vessel, keeping the suture within the lumen. Gently fix the aorta near the tied end with forceps, and then pull the surgical sutures slowly to reverse the aorta until the endothelial surface of the aorta is exposed.
Wash the endothelial surface of the aorta three times with 10 milliliters of fresh washing buffer per wash and place the aorta into a 15-milliliters centrifuge tube. Cover the sample with 10 milliliters of 37 degrees Celsius-warmed 0.005% collagenase IV digestive solution, and incubate the tissue at 37 degrees Celsius for 15 minutes.
At the end of the incubation, transfer the entire tube contents into a 100-millimeter culture dish and arrest the digestion with 10 to 15 milliliters of pre-cooled stopping buffer. Using a cell scraper, gently remove the aortic endothelial cells from the inside surface of the vessel, and wash the vessel three times with 5 milliliters of washing buffer per wash.
After the last wash, transfer the supernatant into a 50-milliliter centrifuge tube, and wash the bottom of the culture dish two times with 5 milliliters of fresh washing buffer per wash, pooling the washes in the 50-millimeter tube.
Collect the cells by centrifugation, and discard all but the last 10 milliliters of supernatant. Add 20 milliliters of washing buffer to resuspend the pellets, taking care to avoid bubbles, and collect the cells with another centrifugation.
Resuspend the pellet in 1 milliliter of cell culture medium for counting, and plate the cells at a 1 x 10 to the 6 cells per 25 centimeters squared flask concentration. Then, place the cells in the cell culture incubator at 37 degrees Celsius and 5% carbon dioxide, refreshing the medium every 2 to 3 days.
