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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. Retighten the cap. Quickly transfer the ampoule to a 37oC 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. Pipette the whole content of the ampoule into a sterile tube (eg 15ml capacity). Then slowly add 5ml prewarmed 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. Transfer the appropriate volume of cell suspension to achieve the cell seeding density recommended on the cell line data entry.

    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 precentrifugation 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 (eg for a cell based assay), rather than subcultured, it may be advisable to perform a precentrifugation step to remove cryoprotectant.

    For suspension cell lines*: A precentrifugation step to remove cryoprotectant is recommended ie 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 cryoprotectant is essential. Rapidly thaw the frozen ampoule in a water bath at 37°C for 1-2 minutes. Transfer the contents to a centrifuge tube and slowly add 5-10ml of prewarmed growth media+. 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 resuspended with media supplemented with 20% FBS in the early critical stage of culture establishment immediately post resuscitation.


 

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