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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.:
Microtiter wells (20)
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).
Scientific Products/ Baxter Healthcare/ Cardinal Health (Surgical Supplies)
Phone # 1-800-964-5227/ 1-800-964-5819
Cat. #2F7154, Lactated Ringer's Irrigation Solution
Vernon Hills, IL
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
Owens and Minor (Surgical Supplies)
Biowhittaker/ Cambrex (Liquid Reagent Supplies)
8830 Biggs Ford Rd., P.O. Box 127
Walkersville, MD 21793-0127
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
Cat. #4197, Type I Collagenase, Crude (5 gm/btl)
M.A. Gimbrone (11/74)
K. C. (11/04)