Core Facilities
Cell Biology > Protocols
Harvesting
Endothelial Cells From
Human Umbilical Cord Vessels
1. Obtain a fresh (less than
24 hours old) umbilical cord in a sterile container.
2. Open sterile cord kit mask and
sterile gloves (see umbilical cord kit contents).
3. Rinse the exterior of the cord
with lactated Ringer's irrigation solution at 37°C
and inspect. Discard if:
a) cord length is less than 12 cm, or
b) clamp marks are less than 12 cm apart.
4. Excise any clamp marks, crushed
areas, or segments pierced by blood sampling needles;
cut 1-2 cm off at each end, cleanly, with a #11 scalpel
blade.
5. Cannulate one end of the umbilical
vein (or artery); normal umbilical cords have two (2)
arteries - smaller and thicker walled, usually in spasm
because of a generous smooth muscle coat; and one (1)
vein - easily dilated to a very large, patulous opening.
Use 2-0 silk suture to tie the cord cannulae or stopcocks
in place (see umbilical cord kit contents).
6. Use 50 cc syringes to flush the
vessel with 200-300 cc of Ringer's to remove any residual
cord blood. (If an adherent internal thrombus is discovered
at this point, the damaged area should be discarded).
7. Carefully cannulate the other
end of the vessel; avoid contaminating the vessel lumen
or cannula tip with red blood cells.
8. Flush through from this end with
100 cc of Ringer's to remove the last traces of red
blood cells; allow the vessel to drain by gravity. Do
not introduce air into the lumen.
* 9. Fill the vessel from the bottom-up
with 0.1% collagenase solution (see * at end of protocol),
pre-warmed to 37°C. Allow a few drops of the dilute
initial effluent to spill over and then close the stopcocks
with tension in vessel. (Fluid-filled sausage.) Do not
use old collagenase solution if a precipitate appears
in the stored liquid (cytotoxic).
10. Place the cord in a sterile jar
(screw-cap, wide-mouth) containing enough 37°C Ringer's
to cover the cords. Incubate in a 37°C water bath
for 10 minutes. Adjust the incubation time to give the
best viability; 10-15 minutes is the normal range.
11. To harvest the endothelial cells,
use 2 syringes to perfuse the cord with a volume of
Hank's Balanced Salt Solution (HBSS, calcium- and magnesium-free
at 37°C) equal to the volume of collagenase solution
contained in the cord; flush from one syringe through
the cord into the other syringe. Repeat the perfusion
about 6 times.
12. Collect the initial effluent
into sterile syringes by gravity drainage and gentle
suction. Air may be used at this step to evacuate the
cell suspension from the cord, unless a subsequent smooth
muscle cell harvest is planned. DO NOT collect effluent
that seeps back into the lumen after the initial evacuation;
it may contain other cell types.
13. Transfer the effluent to a sterile,
conical, siliconized glass centrifuge tube (or plastic
equivalent).
14. Centrifuge the cell suspension
at 4°C in a swinging-bucket rotor at 200g x 5 minutes.
15. Aspirate the supernatant with
a sterile pasteur pipette, under a laminar flow sterile
hood, leaving a small white pellet in the tube.
16. Gently resuspend the cell pellet
in culture medium. The resuspension volume depends on
cell pellet size, and choice of culture flask, e.g.:
CORD LENGTH |
MEDIUM
VOLUME |
FLASK |
20 cm |
5 cc |
T-25 (1),
Microtiter wells (20) |
80 cm |
10 cc |
T-75 (1),
T-25 (3)
Microtiter wells (80) |
17. Incubate at 37°C,
in a 5% CO2-air atmosphere. Change the medium within
12-24 hours of initial plating; thereafter, at 48-72
hour intervals. Examine the cultures daily for growth
using an inverted phase-contrast microscope.
18. Disinfect all effluents, instruments
and work surfaces with a 10% - 15% solution of bleach
or other disinfectant recommended by your institution
for inactivating biohazardous material.
19. Enter cord data and culture observations
in a log book.
UMBILICAL
CORD KIT CONTENTS
Wrap everything, except
the first 3 items, together in a metal tray and autoclave
with the date on wrapper.
- 2 special cord cannulae
with 3-way stopcocks/per cord, sterilized
(May substitute 2 sterile 3-way stopcocks with 1 male
port & 2 female Luer-Lok ports)
- 50cc plastic syringes
x3
- 1 #2-0 silk ligature
pack, sterile
- 1 #11 scalpel blade
- 2 #14 gauge long, blunt
needles (optional, for withdrawing sterile reagents
from bottles)
- gauze sponges (4x8)
- 1 wide-mouth, screw
cap jar
- 1 each hemostat, fine
tooth forceps, scissors, #3 scalpel handle
- 1 each glass beakers:
250, 600 ml (to hold Ringer's, etc. during procedure)
NEEDED
SEPARATELY (suppliers listed below)
Supplies: sterile gloves,
"Barrier Field" drape, masks, goggles/faceshields,
siliconized centrifuge tubes, labcoats;
Solutions: Lactated
Ringer's Irrigation Solution, Collagenase, Nutrient
Medium of 20% heat-inactivated Fetal Bovine Serum in
Medium 199 (v/v), supplemented with 1:100 L-Glutamine
(200mM) and 1:250 penicillin/streptomycin solution (25000
U/ml plus 25,000 mcg/ml), Hank's Balanced Salt Solution
(w/phenol red, w/o calcium or magnesium).
SUPPLIERS
Scientific Products/ Baxter Healthcare/
Cardinal Health (Surgical Supplies)
Columbia, MD
Phone # 1-800-964-5227/ 1-800-964-5819
Cat. #2F7154, Lactated Ringer's Irrigation Solution
Cole-Parmer (Stopcocks)
Vernon Hills, IL
Phone: 1-800-323-4340
Cat. #A31200-80, autoclavable 3-way Luer-Lok stopcock
(Sheild over male port must be cut off before use. Order
2 per cord harvested.)
Allegiance (Surgical Supplies)
McGraw Park, IL
Phone: 1-800-964-5819
Owens and Minor (Surgical
Supplies)
Richmond, VA
Phone: 1-800-949-2508
Biowhittaker/ Cambrex (Liquid
Reagent Supplies)
8830 Biggs Ford Rd., P.O. Box 127
Walkersville, MD 21793-0127
Phone: 1-800-638-8174
FAX: 301-845-8338
Cat. #17-513Q, Dulbecco's PBS (IL)
Cat. #12-118F, Medium 199 (500 ml)
Cat. #10-543F, Hank's Balanced Salt Solution (500 ml)
Cat. #17-605E, L-Glutamine (100ml)
Cat. #17-719R, Penicillin/Streptomycin Solution (25,000U/ml
and 25,000 mcg/ml respectively, in 4.5ml)
Worthington Biochemical Corp. (Enzyme Supplier)
Halls Mill Rd.
Freehold, NJ 07728
Phone: 1-800-445-9603
FAX: 1-800-368-3108
Cat. #4197, Type I Collagenase, Crude (5 gm/btl)
M.A. Gimbrone (11/74)
K. C. (11/04)
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