Core Facilities
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:
Arena's Slaughterhouse
Hopkinton, MA
508-435-3673
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
Boston, MA
7/27/78
(5/19/00) |
