The method is a highly effective method for isolating endothelial cells from miniature pig. And the cells can be expanded too, investigate the immune and coagulation response in xenotransplantation. The main advantage of technique is that it is not only easy, highly effective to isolate highly purified endothelium cells from pig, but also better for keeping other pAECs when passaged.
After confirming a lack of response to pain reflex in the anesthetized pig, wash the porcine abdominal wall three times with 75%medical alcohol, and one tincture of iodine. After the last wash, use a scalpel to make an abdominal incision to expose the inferior vena cava, and use a five milliliter syringe to inject 5000 units of heparin sodium into the inferior vena cava. After five minutes, insert a catheter into the abdominal aorta, and use a scalpel to incise the diaphragm.
With the help of a second investigator, remove the left ventricle and use bone forceps to excise the ribs to expose the heart and lungs. Locate the aorta posterior to the heart and lungs, and clamp the ends. Use scissors to excise the aorta, and wash it one time with pre-cooled washing buffer.
Remove any excess tissue around the aorta with sterile forceps and scissors, and place the vessel into a 50 milliliter centrifuge tube containing fresh pre-cooled washing buffer for transfer to the laboratory. For porcine aortic endothelial cell isolation, transfer the pig aorta into a 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 suture 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 milliliter 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 five 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 five milliliters of fresh washing buffer, per wash, pulling the washes in the 50 milliliter 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 pellet, taking care to avoid bubbles, and collect the cells with another centrifugation.
Resuspend the pellet in one milliliter of cell culture medium for counting, and plate the cells at a one times ten to the six cells per 25 centimeters squared flask concentration. Then place the cells in the cell culture incubator at 37 degrees Celsius in 5%carbon dioxide, refreshing the medium every two to three days. After isolation, the endothelial cells are inspected on day zero, one, and two, and after passages one and two for contamination and their morphological evaluation.
Flow cytometric analysis using an anti-CD31-FITC antibody indicates a typically greater than 97%endothelial cell marker positive population on day three of culture, that is maintained at an over 90%purity even after two passages. The endothelial surface of the aorta was scraped in only one direction, with a cell scraper, for less than 10 times, and it was not compressed during the process of scraping. Best answer, optimized method of pAECs isolation, we can use purified pAECs to perform individual experiments and investigate the immune rejection and coagulation response in xenotransplantation.
