How to Start Your Culture: Thawing Frozen Cells

Ensuring gentle thawing of frozen cells is critical to maintaining their viability and functionality. In this article, we will highlight the cell thawing process, discuss common challenges, and provide recommendations for achieving optimal results.

Preparation of Thawing Cells

When taking out the cryopreservation tube, take personal protection and wear gloves.
Ensure the operating table is clean and sterile to avoid unnecessary contamination that endangers the health of cells
Prepare the experimental equipment to be used, check the water bath temperature, centrifuge and centrifuge tube status, and sterile disposable gloves.
Preheat the media to be used in advance.

Controlled Thawing

Take the cells in the cryopreservation tube out of the frozen storage place, and take them out quickly to avoid the impact of temperature difference on the cells. Cryovials can be thawed in sterile disposable gloves to avoid contamination. Frozen cells should be immediately placed in a 37°C water bath for thawing after being taken out. The step of thawing should be fast and should be completed within 1 minute, which is the key to recovering cells.
Wipe the outside of the cryovial with 70% ethanol and transfer it into a sterile operating table, and transfer the pre-warmed medica into it to slowly dilute the thawed cells. Use a pipette gun to slowly draw the liquid in the bottle into a 15ml centrifuge tube, then add an appropriate amount of culture media, and mix gently.
Next is centrifugation: centrifugation speed and duration vary by cell type. After centrifugation, discard the supernatant, add fresh media to resuspend the cells, and transfer to a suitable culture container and culture environment.

Precaution

Be careful of the damage caused by low-temperature when taking out the frozen cells, and wear antifreeze gloves.
It is very important to resuscitate the cells quickly, try not to exceed one minute.
After thawing, try to maintain high density to improve cell survival rate.
Pay attention to aseptic operation and prevent cell contamination, which can help the cells to maintain a good state.

Troubleshooting

We have listed some potential problems and possible solutions that may help you troubleshoot your cell culture experiments.

a) No viable cells after thawing

Cells were stored incorrectly: Obtain new stock and store in liquid nitrogen. Keep the cells in liquid nitrogen until thawing.

Cells were thawed incorrectly: Follow procedures for thawing cells exactly as recommended by the supplier. Be sure to thaw frozen cells quickly, but dilute them slowly with pre-warmed growth media before plating.

Thawing media is not correct: Use the media recommended but the supplier. Make sure the media is pre-warmed.

Cells are too dilute: Plate thawed cells at high density as recommended by the supplier to optimize recovery.

Cells not handled gently: The freezing and thawing procedures are stressful to most cells. Do not vortex, bang the flasks to dislodge the cells (except when culturing insect cells), or centrifuge cells at high speed.

b) Cells grow slowly

Growth media is not correct: Use pre-warmed growth media as recommended by the supplier.

Poor quality serum in growth media: Use serum from a different lot.

Cells have been passaged too many times: Use healthy cells with low passage-number.

Culture is contaminated with mycoplasma: Discard cells, media, and reagents. Obtain new stock of cells, and use them with fresh media and reagents.

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