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Cell Biology > Protocols
Bovine Aorta Smooth Muscle Explants
1. The procedure is carried out using sterile technique.
2. Remove aortas from freshly slaughtered calves. Place them in a sterile container. Keep aortas on ice during transport. For lengthy transportation, place aortas in a sterile, leak-proof container half-filled with Dulbecco's PBS or other isotonic solution supplemented with 200 U/ml Penicillin and 200 mg/ml Streptomycin. (No liquid is needed for brief trips.)
3. Instruments and solutions are set up on a sterile barrier field:
- 2-3 Tissue-culture treated petri dish (100x20mm) per aorta
- 1 each #22 Scalpel blade and blade holder, Crille forceps
- 2 pair Iris Scissors, 4-4 1/2" straight +/or curved
- 3 Iris Forceps, 4-4 1/2", fine-toothed straight 1x2 teeth
- Sterile Gauze, 8x4 in.
- Additional sterile barrier field
4. An aorta is removed from the container using Crille Hemostats and is placed on an additional sterile barrier field. The aorta is swabbed with 100% Wescodyne solution. The ends are removed with a sterile blade and the aorta is transferred to a fresh barrier field.
5. Using Iris scissors and forceps, remove the fascia. Discard these instruments.
6. Using a clean pair of scissors and forceps, remove sections of the aorta, about 5cm x 2cm (depending on the size of the aorta), being careful not to remove the area with the intercostal vessels.
7. Place the strips in a petri dish until ready to separate the layers.
8. Discard the remaining aorta.
9. Grasp the strip at the narrow end between 2 tissue forceps. Hold the forceps on the adventitial side rigid and use the other pair to carefully peel off the thin intimal layer and media, about 1/4 of the total thickness of the aorta. Discard the adventitia.
10. Place the strip of media with intima back into the petri. The following steps are carried out in a Laminar Flow Hood.
11. Add 10%CS in DMEM to the petri. Cut the strip into small pieces using sterile Iris Scissors.
12. Place the small pieces (explants) intimal side up (shiny-side up) into new petri dishes. Use sterile forceps.
13. Add medium (10%CS in DMEM) to the new petri dishes in a volume sufficient to surround the explants but not to cover them. Don't flood the dishes. This allows for the explants to adhere to the petri surface overnight. The next day add an additional 5-8cc of medium. Three to four days later, exchange the medium. Re-feed the cultures every Monday, Wednesday and Friday from then on.
14. Explant growth is visible within 6-8 days. Plates are 75-80% confluent in 2 weeks. Explants can be removed. The cultures may be used before reaching confluency. It is easier to trypsinize and passage the cells before they become too dense and/or piled-up.
Source of Aorta:
John - deliveries 1-2:30 pm. tues.and wed.
Mass Pike to 11A
Right at toll, southbound
Take exit 21A (2nd exit)
Follow to town center.
Bear right at statue, past school
About1 mile down the road on left.
Dr. Michael A. Gimbrone, Jr.
Sheila A. Cruise
Brigham & Women's Hospital