Freezing Tissue Culture Cells

Cells are typically frozen in some form of cryoprotectant to help prevent membranes from rupturing by the formation of ice crystals during the freezing process. It is generally believed that the more gradually the temperature of the cells is lowered, the higher is the viability. The endpoint for freezing tissue culture cells is always liquid nitrogen, i.e. -165C or colder. Below this temperature essentially all processes have stopped and the cells can be maintained for years with no decrease in viability. Above this temperature, cellular viability gradually (or rapidly) degrades in a cell-type dependent manner. This means that the -80C temperature of an standard ultra-low freezer is NOT acceptable for long-term storage of tissue culture cells.

1. treat log-phase growing ~50% confluent adherent monolayer cultures with trypsin/EDTA solution until cells are mostly detached, add back two volumes of complete medium and pipette up and down to disperse clumps and ensure complete detachment
   
   • skip this step when dealing with non-adherent (suspension) cultures
     
2. transfer cells to a 15 ml conical centrifuge tube. centrifuge at 200 g for 5 minutes in a clinical centrifuge to pellet the cells
   
3. carefully aspirate off the medium to leave the cell pellet. resuspend the cell pellet by repeated gentle pipetting in 1 ml of:
   
   • 95% complete medium
     
   • 5% DMSO (dimethyl sulfoxide)
     
4. transfer cells suspended in the cryosolution above to a freezing vial (there are vials specifically designed for storage at liquid nitrogen temperatures)
   
5. put the cryovial with cells into the vapor phase of a liquid nitrogen freezer. store indefinitely in the vapor phase. transfer to the liquid phase after complete freezing if absolutely necessary due to freezer-space limitations [not recommended]
   
   • do NOT put the vial directly into the liquid phase. the vapor phase will allow for automatic slow cooling as the heat capacity of nitrogen vapor is very low
     

see also: ThawingTissueCultureCells

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Comments

• there are other cryoprotectants than DMSO. some people prefer high (30%+) serum
  
• in general, specially designed "slow-cooling" freezer boxes are not necessary so long as the vials are in the vapor phase of the liquid nitrogen freezer
  
• storage of vials in the liquid phase of liquid nitrogen is not recommended due to possible issues with contamination and "exploding vials" when liquid nitrogen leaks past the seal of the vial. there is no difference in the effectiveness of long-term storage below -165C, so there is no advange to liquid phase (-196C) over vapor phase (-185C) storage