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Animal cell culture: cell cryopreservation and resuscitation
Cryopreservation is a widely used technique for long-term cell storage. By freezing cells in liquid nitrogen at -196°C, they can be temporarily put into a dormant state while maintaining their original biological characteristics. This allows researchers to revive the cells when needed for experiments. Storing cells cryogenically also helps prevent contamination or loss due to unexpected issues during culture, making it an essential method for cell conservation. Additionally, cryopreserved cells can be easily transported, shared, or exchanged between laboratories.
When freezing cells, a cryoprotective agent such as 5–15% glycerol or dimethyl sulfoxide (DMSO) is added to the culture medium. These agents lower the freezing point of the solution and help reduce ice crystal formation by allowing water to slowly exit the cells during the freezing process. The "slow freezing and rapid thawing" method is commonly used to maximize cell viability. The freezing rate typically starts at -1 to -2°C per minute, accelerates below -25°C, and then the cells are transferred directly into liquid nitrogen at -196°C. For thawing, the frozen vials are quickly placed in a 40°C water bath to ensure minimal damage to the cells.
**Materials required:** Cryovials, centrifuge tubes, cell culture supplies, 500 mL beakers, tape, enamel cups, and 75% alcohol-soaked cotton.
**Reagents needed:** Culture medium (RPMI-1640 or DMEM), fetal bovine serum, 0.25% trypsin solution, DMSO or glycerol, 0.5% trypan blue, and liquid nitrogen.
**Equipment required:** A 4°C refrigerator, a -20°C freezer, a -70°C freezer, a liquid nitrogen container, a micropipette, a water bath, and a centrifuge.
**Step-by-step procedure for cell cryopreservation:**
1. Detach the cells using trypsinization, then wash them with fresh culture medium.
2. Centrifuge the cells at 800 rpm for 5 minutes to pellet them, then discard the supernatant.
3. Resuspend the cells in a cryopreservation medium containing 10% DMSO or glycerol mixed with 90% culture medium.
4. Adjust the cell concentration to approximately 3 × 10ⶠcells/mL, and increase the serum concentration to 20% if necessary.
5. Transfer the cell suspension into cryovials, label them clearly with details such as cell type, date, and freezing conditions.
6. Freeze the vials gradually: first at 4°C for 30 minutes, then at -20°C for 1.5 to 2 hours, followed by -70°C for 4 to 12 hours before final storage in liquid nitrogen.
**Procedure for cell resuscitation:**
1. Heat an enamel cup with tap water to around 40°C.
2. Retrieve the frozen vial from liquid nitrogen and immediately thaw it in the warm water.
3. Wipe the vial’s cap with 75% alcohol and open it carefully.
4. Centrifuge the cell suspension, then wash the cells with serum-free medium.
5. Resuspend the cells in 3 mL of fresh culture medium and transfer them to a small flask.
6. Monitor the cells daily for growth and viability. If many cells appear dead, replace the medium the next day. Once the cells reach confluence, they can be subcultured as needed.
This detailed protocol ensures that cells remain viable after cryopreservation and can be successfully revived for future experiments.