Thawing Human Dermal Fibroblasts (Neonatal or Adult)

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



  1. Warm FGM™-2 medium and HBSS 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 dermal fibroblasts (neonatal and adult) have a recommended cell seeding density of 2,000-3,500 cells/cm2. The 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;
    1. Number of flasks = number of cells/cell seeding density/flask area in cm2
    2. 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 1.5 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. Add 500 µL of HBSS into a sterile centrifuge tube;
  9. Add the thawed cell suspension to the centrifuge tube from step 8;
  10.  Pipette up and down to mix cells;
  11.  Follow this protocol to count cells;
  12.  You will need to add 1 mL of cell suspension to each media-containing flask, so add the remaining amount of media to your cell suspension tube (since you resuspended it in 1-5 mL for counting) to have a total volume of nflasks;
    1. Example: If you resuspended your cells in 1 mL and you are replating them in 5 flasks, add 4 mL to complete the suspension volume necessary to be able to add 1 mL of cell suspension per flask.
  13.  Add 1 mL of cell suspension to each media-containing flask;
  14.  Gently rock flasks;
  15.  Label your flasks. It is good practice to include the following information;
    1.  Your initials and date of passage (e.g.: if your initials are YN, and the date is MM/DD/YYYY, label it as YNYYYMMDD)
    2. Cell line (HDFN-Neo or HDFN-Ad) and passage number (if your vials were Pn, they will now be Pn+1).
  16.  Place flasks in the incubator;
  17.  After 36-48 h, change cell medium by following this protocol;
  18.  Feed cells every two to three days;
  19.  Passage cells following this protocol.

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