Cell Biology > Protocols
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
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
16. Dip the applicator
into the tube of medium and swirl it gently to dislodge
the harvested cells. (Use separate tubes and swabs for
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
19. Discard the supernatant
and re-suspend the pellet in 5-6ml of the 20% calf serum
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
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,
- Calf Serum (e.g. GIBCO, Cat. #16170-086)
- L-Glutamine (Biowhittaker, Cat. #17-605E)
- Concentrated Penicillin/Streptomycin Solution (Biowhittaker,
- Collagenase, Type I, crude (Worthington Biochemicals,