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Cell Biology > Protocols
Bovine Aorta Endothelial Cell Protocol
1. The procedure is carried out using sterile technique.
2. Aortas are handled separately to avoid cross-contamination. Aortas should be kept refrigerated until they are used, preferably within 4 to 6 hours of being taken from the animal.
3. An aorta is laid on a sterile barrier. Wescodyne, or another iodine-based detergent/ disinfectant, is applied to the exterior of the aorta by means of a squeeze bottle. Care should be taken to avoid the open ends of the aorta since the disinfectant will kill the endothelial cells if it touches the interior surface of the aorta.
4. Transfer the aorta to a different, clean section of the sterile barrier or to a piece of sterile gauze and remove the fascia as thoroughly as possible, by blunt dissection using an expanding motion with the dissecting scissors to peel away layers of adventitia, etc.
5. With a #22 scalpel blade, cut off and discard the ends of the aorta.
6. With a pair of blunt/sharp scissors cut the aorta open longitudinally. Adson-toothed forceps may be used to steady the tissue.
7. With blunt/sharp scissors remove the portion of the aorta wall containing the intercostal vessels, taking care not to damage the luminal surface of the rest of the aorta.
8. Excise any areas which have been contaminated with Wescodyne.
9. Rinse the segment by immersing it several times in a 250cc beaker of lactated Ringer's irrigation solution, or other balanced salt solution. If several aortas are being processed at the same time, cleaned segments may be held in a sterile container of cold (4°C) Ringer's while subsequent aortas are cleaned.
10. Transfer clean aorta segments to individual 10 cm petri dishes, keeping the luminal surface uppermost. Continue the procedure in a laminar-flow sterile hood.
11. Drip a sterile solution of 0.1% collagenase from a pasteur pipet onto the luminal surface of each aorta, being very careful not to let the collagenase come into contact with the edges of the segment in order to avoid contact of the enzyme with the exposed smooth muscle layers.
12. Incubate the segments at room temperature for approximately 10 minutes.
13. For each aorta, fill 1 sterile 15cc centrifuge tube with 5-10 ml of sterile Dulbecco's Modified Eagle's medium supplemented with 20% heat-inactivated calf serum,
2mM L-Glutamine, and an antibiotic solution of 100 U/ml penicillin and 100 mg/ml streptomycin.
14. Moisten sterile cotton-tipped applicators (cotton swabs with wooden handles) with this medium, using a separate swab for each aorta.
15. Starting 2-3mm from the upper edge of the aorta, touch the applicator to the luminal surface and draw it longitudinally down the segment using gentle to moderate pressure and rotating the swab slowly between the thumb and forefinger as it progresses.
16. Dip the applicator into the tube of medium and swirl it gently to dislodge the harvested cells. (Use separate tubes and swabs for each aorta).
17. Repeat Steps 15 and 16, moving the applicator to an adjacent BUT NOT OVER-LAPPING area. On no account should any portion of the surface be swabbed more than one time, as underlying smooth muscle cells would then contaminate the harvest.
18. Centrifuge the tube in which the applicator has been rinsed and the cells collected for 5-10 minutes in a swinging bucket rotor at 1000rpm (200G) at 4°C. If several aortas are being harvested, the tubes from the completed aortas may be held in an ice bucket to prevent excessive proteolysis while subsequent aortas are harvested, permitting all the samples to be centrifuged simultaneously.
19. Discard the supernatant and re-suspend the pellet in 5-6ml of the 20% calf serum medium.
20. Plate this cell suspension in a 25cm2 tissue culture flask and incubate the culture in a 5% C02 atmosphere at 37°C overnight. Make a separate flask for each tube of cells to avoid cross-contamination.
21. On the following day, wash away debris and unattached cells with a balanced salt solution such as Hank's w/o Ca2+ & Mg2+, and re-feed with 20% calf serum medium.
22. The cultures are to be re-fed at 48-72 hour intervals (e.g., Monday, Wednesday, Friday) when they begin to show evidence of cell division, typically within 2 to 4 days.
23. Cultures may be weaned to 15% calf serum at confluence, and may be further weaned to a maintenance medium containing only 10% calf serum either by holding the primary culture at confluence or by reducing the serum concentration when the first sub-culture of cells reaches confluence.
- Dulbecco's Modified Eagle's Medium, low-glucose (GIBCO, Cat. #12320-032)
- Calf Serum (e.g. GIBCO, Cat. #16170-086)
- L-Glutamine (Biowhittaker, Cat. #17-605E)
- Concentrated Penicillin/Streptomycin Solution (Biowhittaker, Cat. #17-719R)
- Collagenase, Type I, crude (Worthington Biochemicals, Cat. #4197)