Porcine Aortic Endothelial Cell (pAEC) Isolation: An Enzyme-based Method to Isolate Endothelial Cells from Harvested Porcine Abdominal Aorta
Transkript
The aorta – the major artery carrying oxygenated blood from the heart to the body – is composed of three layers or 'tunics': an inner intima, a middle media, and an outer adventitia. Endothelial cells or ECs are present in the intima as a thin, continuous monolayer over a supporting basement membrane consisting of structural proteins, primarily collagen.
To isolate ECs, begin with a harvested porcine aorta in a culture dish. Excise its ends and any surrounding adherent tissue to prevent non-endothelial cell contamination. Wash the aorta with buffer containing antibiotics to inhibit microbial growth.
Pass a sterile surgical suture through the aorta and tie one of its ends, keeping the suture within the lumen. Secure the tied end and retract the suture to invert the aorta completely. This exposes the EC-containing intima to enzymatic digestion. Treat the aorta with pre-warmed, low-concentrated collagenase solution for optimal enzyme activity and incubate.
Collagenase breaks down the collagen proteins present in the basement membrane underlying the ECs, initiating their dissociation. Post-incubation, add chilled buffer to inactivate the collagenase enzymes.
Gently scrape the aortic intimal surface and rinse it with buffer to release the ECs into solution. Centrifuge this solution to pellet the ECs. Resuspend the pellet in suitable medium. Seed the cell suspension in a culture flask and incubate. The ECs adhere to the flask bottom and proliferate.
