Cell Freezing Protocol

Preserving cells through freezing is essential for research, veterinary medicine and animal breeding because it allows cells to be stored for long periods of time. The freezing method selected, however, must reduce or prevent damage to the cell. The cryopreservation agent and method can depend on cell type.

Preparation

• Cells should be frozen when they are healthy and actively growing with a 90 percent viability. Care should be taken on handling and high-speed centrifugation and vigorous pipetting or suctioning should be avoided to maintain viability and prevent cellular damage.

Cryoprotection

• A cryoprotection agent is used as a replacement for intracellular water. The agent prevents ice crystals and dehydration occurring in the cells. Sterilized glycerol and sterilized dimethylsulfoxide are often used at a concentration of 5 percent to 15 percent (volume per volume) diluted in either fetal calf serum or growth media, depending on cell type.

Freezing and Storage

• Cells in the freezing mixture should be cooled slowly. If a programmable freezer is not available, cryovials can be wrapped in tissue or placed in styrofoam, which may slow the freezing process. Cells should be kept at minus 80 degrees Celsius for 24 hours and then transferred to liquid nitrogen for long-term storage.

Defrosting

• Frozen cells should be thawed quickly. A water bath at 37 degrees Celsius can be used. Care should be taken with cells stored in liquid nitrogen, however, as liquid nitrogen can enter the vials and can explode on thawing. The freezing mixture should be diluted in growth media. Cell viability should be assessed with the dye trypan blue and the cells cultured as normal.