Thawing Human Umbilical Vein Endothelial Cells (HUVECs)

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Thawing cells properly is crucial to cell culture health. Follow this protocol to ensure your cryopreserved human umbilical vein endothelial cells will have an optimal yield for your experiments.



  1. Warm EGM™-2 medium to 37˚C in the water/bead bath;
  2. Remember to spray everything down with 70% ethanol and wipe it with Kimwipes before moving into the BSC;
  3. Check the number of cells that are in the cryopreserved vial and prepare the appropriate number of flasks with the corresponding amount of warm cell medium. Human umbilical vein endothelial cells have a recommended cell seeding density of 2,500 cells/cm2. Amount of media will also vary by flask size, but it is recommended to use 0.2 mL of media per cm2. Find useful formulae below;
    • Number of flasks = number of cells/cell seeding density/flask area in cm2
    • Amount of media = (flask area in cm2)*0.2 mL
  4. Place these media-containing flasks into the incubator for 30 minutes before adding cells;
  5. Remove cell vial from liquid nitrogen and submerge it in the water/bead bath up to the cap for no longer than 2 minutes;
  6. Remove vial when its sides are thawed but its center remains frozen;
  7. Spray down all media and pipets with 70% ethanol, wipe them with Kimwipes and move them into the BSC;
  8. Mix the vial cell suspension by pipetting up and down;
  9. Pipette corresponding volumes to the flasks you prepared on step 3;
  10.  Gently rock flasks;
  11.  Label your flasks. It is good practice to include the following information;
    •  Your initials and date of passage (e.g.: if your initials are YN, and the date is MM/DD/YYYY, label it as YNYYYMMDD)
    • Cell line (HUVECs) and passage number (if your vials were Pn, they will now be Pn+1).
  12.  Place flasks in the incubator;
  13.  After 16-24 h, change cell medium by following this protocol to remove DMSO;
  14.  Feed cells every other day;
  15.  Passage cells following this protocol.

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