Most cell cultures can be stored for many years, if not indefinitely, at temperatures below -130°C (cryopreservation). ATCC has recovered cells from cultures cryopreserved for more than 40 years. The many advantages of cryopreservation far outweigh the required investment in equipment and reagents. These advantages include:
- Generation of safety stocks to ensure against loss of the culture from equipment failures or
contamination by microorganisms or other cell lines.
- Elimination of the time, energy, and materials required to maintain cultures not in immediate use.
- Preservation of cells with finite population doublings (that will ultimately senesce).
- Insurance against phenotypic drift in the culture due to genetic instability and/or selective pressure.
- Creating a standard reagent to be used for a series of experiments.
As the cell suspension is cooled below the freezing point, ice crystals form and the concentration of the solutes in the suspension increases. Intracellular ice can be minimized if water within the cell is allowed to escape by osmosis during the cooling process. A slow cooling rate, generally -1°C per minute, facilitates this process. However, as the cells lose water, they shrink in size and will quickly lose viability if they go beyond a minimum volume. The addition of cryoprotectant agents such as glycerol or dimethylsulfoxide (DMSO) will mitigate these effects.
The standard procedure for cryopreservation is to freeze cells slowly until they reach a temperature below -70°C in medium that includes a cryoprotectant. Vials are transferred to a liquid-nitrogen freezer to maintain
them at temperatures below -130°C.
The recovery of cryopreserved cells is straightforward: Cells are thawed rapidly in a water bath at 37°C,
removed from the freeze-medium by gentle centrifugation and/or diluted with growth medium, and
seeded in a culture vessel in complete growth medium.
There are numerous factors which affect the viability of recovered cells. Modify the procedure for each cell
line to attain optimal cell viability upon recovery. Some of the critical parameters for optimization include
the composition of the freeze medium, the growth phase of the culture, the stage of the cell in the cell cycle,
and the number and concentration of cells within the freezing solution.
ATCC provides information on cryopreservation for all cell lines on the Product Sheet. Most ATCC cell lines
are frozen with a cryopreservation medium consisting of 5% DMSO and complete growth medium.