Protocol: Resuscitation of Frozen Cells

It is important to handle frozen ampoules with care; wear a laboratory coat, full protective face mask and gloves. On rare occasions ampoules may explode on warming due to expansion of trapped residual liquid nitrogen.

1. In a microbiological safety cabinet, hold a tissue soaked in 70% alcohol around the cap of the frozen ampoule and turn the cap a quarter turn to release any residual liquid nitrogen that may be trapped. Re-tighten the cap. Quickly transfer the ampoule to a 37^oC waterbath until only one or two small ice crystals, if any, remain (1-2 minutes). It is important to thaw rapidly to minimise any damage to the cell membranes.
   
   Note: Do not totally immerse the ampoule as this may increase the risk of contamination.
   
   
2. Wipe ampoule with a tissue soaked in 70% alcohol prior to opening.
   
   
3. Slowly pipette the whole content of the ampoule into 5ml pre-warmed medium that has already been supplemented with the appropriate constituents. Determine the viable cell density using trypan blue stain, a haemocytometer and an inverted microscope to count the cells or equivalent cell counting method.
   
   For adherent cell lines: Adjust the volume of the medium, and if necessary the flask size, to achieve the cell seeding density recommended on the cell line data sheet. A pre-centrifugation step to remove cryoprotectant is not normally necessary as the first media change will remove residual cryoprotectant If it is, then this will be specified on the data sheet. If the cells are to be used immediately (e.g. for a cell based assay), rather than subcultured, it may be advisable to perform a pre-centrifugation step to remove cryoprotectant.
   
   For suspension cell lines*:A pre-centrifugation step to remove cryotprotectant is recommended i.e. pellet the cells by centrifugation at 150 x g for 5 minutes and resuspend the cell pellet in fresh medium using the appropriate volume to achieve the correct seeding density.
   
   
4. Incubate flasks at the temperature and CO₂ level recommended on the data sheet. If a CO₂ fed incubator is used the flask should have a vented cap to allow gaseous exchange.

*Important note regarding hybridoma cultures: When recovering hybridoma cultures from frozen it is not unusual for growth initially to be slower than expected and there may be an observed decrease in viability. Establishment of an actively proliferating culture may take up to 2 weeks.

On resuscitation a centrifugation step to remove the cryo-protectant is essential. Rapidly thaw the frozen ampoule in a water bath at 37°C for 1-2 minutes. Slowly pipette the whole content of the ampoule into 5ml pre-warmed medium that has already been supplemented with the appropriate constituents⁺. Remove a sample for counting. Centrifuge at 100 x g for 2-3 minutes to pellet cells and seed at a relatively high density of 5-7 x 10⁵ cells/ml. Place culture flask flat and observe regularly until viable proliferating cells are seen.

+Often hybridoma cultures may benefit from being re-suspended with media supplemented with 20% FBS in the early critical stage of culture establishment immediately post resuscitation.

Related Links:
Cell Counting Using a Haemocytometer