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 a 25cm² flask containing 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. 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. If it is, then this will be specified on the data sheet.
   
4. Incubate at the temperature and CO₂ level recommended on the cell line data sheet. If a CO₂ fed incubator is used the flask should have a vented cap to allow gaseous exchange.
   
   
   
   Related Links:
   Cell Counting Using a Haemocytometer