Preparing HUVEC

Tissue

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.

Protocol

  1. Discard any damaged areas of cord. Check for needle holes and blood clots.
  2. Cut one end of the cord to obtain a fresh cut.
  3. 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.
  4. 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.
  5. Clamp the other end of the vein.
  6. Fill the vein with collagenase solution via the small tube that is joined to the cannula.
  7. Clamp the tube to seal the vein.
  8. Incubate the cord for 30 min at 37°C in a beaker containing PBS.
  9. 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
  10. Centrifuge 5 min at 2,000g
  11. Resuspend in 15 ml EGM-2 medium (endothelial growth medium; Lonza).
  12. Seed cells into a 25 cm flask and place in gassed incubator at 37°C.

Reagents

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

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