Cells should be prepared from freshly obtained umbilical cords. These can be stored in ice-cold PBS in a 250 ml sterile container at 4°C for 6-48 h.
- Discard any damaged areas of cord. Check for needle holes and blood clots.
- Cut one end of the cord to obtain a fresh cut.
- Insert a sterile 8cm piece of silicone tubing (3mm ID, 5mm OD) into one end of the vein and fix it tightly in place with surgical silk.
- Connect a syringe filled with 20-25 ml PBS into the tube. Remove the blood from the vein by slowly flushing the blood vessel and draining the contents. Ensure that no air is introduced into the vessel.
- Clamp the other end of the vein.
- Fill the vein with collagenase solution via the small tube that is joined to the cannula.
- Clamp the tube to seal the vein.
- Incubate the cord for 30 min at 37°C in a beaker containing PBS.
- Remove the clamps and collect the contents of the vein lumen into a 50 ml Falcon (disposable polypropylene) tube. Flush the vessel with 20 ml DMEM/F12 medium
- Centrifuge 5 min at 2,000g
- Resuspend in 15 ml EGM-2 medium (endothelial growth medium; Lonza).
- Seed cells into a 25 cm flask and place in gassed incubator at 37°C.
DMEM/F12 medium: DMEM/F12 medium (50:50) with 10% FBS, 100U/ml penicillin, 100µg/ml streptomycin.
Collagenase: 0.1% collagenase (Type 3; Worthington #CLS3) in DMEM/F12 medium