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Protocol for Thawing Cells

What You Will Need:

  • Vial of cells to thaw
  • 1 centrifuge tube (preferably 15ml)
  • 1 TSB tube
  • Flask(s) for growth
  • Appropriate media
  • Pipettes

Expected Time Requirement:
~30 minutes/vial for full thaw process


  1. Retrieve a vial of cells from liquid nitrogen storage.
    1. Log out the vial on the locator map sheet.
    2. Transport the cells on dry ice.
  2. Put about 10ml of the appropriate media in a sterile labeled centrifuge tube. Media does not need to be warm. Media should not contain antibiotics or selective agents.
  3. Thaw the cells by swishing the vial in a 37 +/-2◦C waterbath.
    1. Do not submerge the vial.
    2. Do not get the gasket wet.
    3. Thaw the vial until a tiny piece of ice remains, do not hold the vial at 37◦C any longer than is necessary.
  4. Spray the vial with alcohol and allow it to dry in the hood. It is important that the vial is dry as any remaining liquid could get into the vial when it is opened, thereby contaminating the cells.
  5. Carefully open the vial. Remove the contents of the vial with a small pipette. Add the volume to the centrifuge tube. Rinse the pipette with the media in the tube and gently mix the cells.
  6. Centrifuge the tube at ~1200 rpm for 5 minutes with the brake off. (this is a gentle spin to pellet the cells. The purpose of this is to wash the DMSO [which is toxic to the cells] away from the cells. It also gives the opportunity to check the sterility of the freeze.).
  7. Remove the media from the pellet and place ~ 2 ml it in a TSB tube. Discard the remainder. Label the tube with the cell line name, and the date. Place the tube in an incubator. This is your sterility check.
  8. Gently resuspend the pellet in fresh media. Put the full volume in the growth vessel desired (usually a T25 or T75) and add media. If possible, use media without antibiotics. As a rule of thumb, select a vessel which will allow seeding at: 1 - 10 x 10^4 cells /cm2.
  9. Label the flask with the cell line name, passage number (increase the passage number by 1 from the passage on the vial. ie. If the vial is labeled p5, label the flask p6), your initials and the date. Incubate the cells in the appropriate incubator.
  10. Fill out the culture sheet with the cell line name and an exact copy of the information on the vial in the upper right corner. If possible, attach the actual label.
    1. Fill out the first line of the culture sheet. There will be no split ratio. Indicate how many vials were thawed and how many vessels (and what size) were seeded.
    2. Specify what method was used for thawing (see notes below)
    3. Fill out the second line with the sterility check information.
  11. Observe the flasks each day for the next several days and write down the observations for each day. These comments will be useful the next time the cell line is thawed.
    1. Be sure to observe the sterility tube for evidence of contamination. This tube is useful because there will be little to no cell debris to contend with. It is not unusual for some cells to make it into the sterility tube; therefore, do not determine sterility based on turbidity, examine the tube under a microscope.
    2. Record the sterility results on the culture sheet at least 7 days post thaw.
  12. A feed may be required as cells often require extra time to reach confluence following thawing. Feed every 2 or 3 days if needed. If cells begin to form "colonies" but are still <75% confluent, split to break up clumps at ~1:1, 1:2, 1:3 as appropriate.
  13. Take a mycoplasma sample (if needed). See Mycoplasma testing Protocol.
  14. Split cells as required.

Frozen, recently thawed cells are very delicate. Handle them gently.

If your cell line is known to be delicate or to have poor viability following freezing, several steps can be taken to improve survivability.

  1. Thaw the cells to a smaller flask, this increases the cell density and often improves cell growth.
  2. Thaw the cells into cold media, spin them and resuspend them in warm media that has been held in the incubator for at least 15 minutes to allow CO2 adjustment.
  3. Thaw the cells and add them directly to a flask of warm and CO2 adjusted media. Incubate. The next day, remove the residual DMSO by aspirating the media off and adding fresh, warm media. Note: This will not give you as direct a look at the sterility of the vial. This has the advantage of removing dead cells from the culture.
  4. Thaw the cells into cold media, bring the final volume to 10ml. Remove 0.1ml of the suspension and perform a cell count. Hold the suspension on wet ice while you count. Spin the cells as described above. Using the final volume of media and cells, calculate and seed at 1 - 10x10^4 LIVE cells/cm2.
  5. If possible, use media without antibiotics. Many selective agents are also antibiotics, check the media recipe carefully. This will improve post thaw survival. Remember to add the selective agent back in later. Carry the cells with selection for at least one passage before harvesting the cells for use. Note that many cells will die when the selective agent is added and seed heavier than usual for that first passage.
  6. Thaw the cells as quickly as possible.