Porcine Aortic Endothelial Cell (pAEC) Isolation: An Enzyme-based Method to Isolate Endothelial Cells from Harvested Porcine Abdominal Aorta

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Isolation of Porcine Aortic Endothelial Cells

1. Under aseptic conditions, remove the pig aorta from the washing buffer, and place it on a 150 mm Petri dish (Figure 1A).
2. Gently cut off the two ends of the aorta and excise excess tissue around aorta with sterile forceps and scissors again (Figure 1B). Wash the outside and inside of the aorta (over the culture dish at room temperature) with 20−50 mL of washing buffer: 1x PBS solution (pH 7.4) with 1% (v/v) Penicillin/Streptomycin.
   NOTE: Try to only cut the area where the clamps were placed during surgery since some ECs were damaged in this area, and remove excess tissues and arterial side branches.
3. Pass a surgical suture through the aorta and then tie one end of the aorta using the surgical suture (5-0, 90cm, Table of Materials). Keep the surgical suture in the inside of the aorta (Figure 1C,D).
4. 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 (Figure 1E−G).
   NOTE: Ensure to fix the aorta near the tied end and do not fix the aorta tightly, or else the aorta cannot be reversed by pulling the surgical suture.
5. Wash the endothelial surface of the aorta with washing buffer 3x (10 mL per wash), and then discard this solution. Place the aorta into a 15 mL centrifuge tube, and cover the aorta with 10 mL of 0.005% collagenase IV digestive solution in the tube (Figure 1H).
   NOTE: Pre-warm the 0.005% collagenase IV digestive solution at 37 °C before digestion.
6. Incubate at 37 °C for 15 min. Place the digested aorta and digestive solution into a 100 mm culture dish and stop the effects of 0.005% collagenase IV by adding 10−15 mL of pre-cooled stopping buffer: Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated FBS and 1% P/S.
   NOTE: The recommended digestion time is between 10 and 20 minutes. Make sure the endothelial surface of the aorta is covered by the stopping buffer.
7. Gently scrape pAECs off the inside surface of the aorta using a cell scraper (Figure 1I). Wash the scraped aorta 3x with washing buffer (5 mL per time). Put the solution from the culture dish into a 50 mL centrifuge tube. Wash the bottom of the culture dish 2x with washing buffer (5 mL per time), and put the solution into a 50 mL centrifuge tube again.
   NOTE: Scrape in one direction gently and do not compress. Do not touch the tissue near the edges and holes. Scraping 5−8x is recommended.
8. Centrifuge the tube at 600 x g for 6 min at 4 °C. Discard the supernatant and leave 10 mL of solution at the bottom of a 50 mL centrifuge tube. Add 20 mL of washing buffer to the 50 mL centrifuge tube and resuspend the pellets. Avoid bubbles during this step.
9. Centrifuge at 600 x g for 6 min at 4 °C. Slowly discard the supernatant. Resuspend the cell pellets with 1 mL of culture medium and mix well.
   NOTE: The obtained average number of ECs per cm aorta is 1.9 x 10⁵ ± 1.4 x 10⁴ (mean ± SD, n = 3).
10. Count the cells with a cell counter. Plate and culture the cells in a vessel without coating any materials according to the cell number. If the cell number is less than or equal to 1.0 x 10⁶, culture cells in a 25 cm² flask. If the cell number is larger than 1.0 x 10⁶, culture cells in several 25 cm² flasks (1.0 x 10⁶ cells per flask). Place the flask in an incubator (without shaking) at 37 °C with 5% CO₂ and replace the medium every 2−3 days.
    NOTE: Some cells are damaged by the cell scraper. A cell viability assay found the percentage of live cells to be 95.7% ± 1.7% (mean ± SD, n = 3). The doubling time of the isolated cells is about 1−2 days.