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Animal cell culture: cell cryopreservation and resuscitation
Cryopreservation is a widely used technique for long-term storage of cells. By freezing cells in liquid nitrogen at -196°C, they can be temporarily put into a dormant state while maintaining their biological characteristics. This allows for future resuscitation and use in experiments when needed. Storing cells cryogenically also helps prevent contamination or loss due to unexpected issues during cell culture, thus serving as an effective method for cell conservation. Additionally, cryopreserved cells can be easily transported, exchanged, or purchased between laboratories.
During the freezing process, a cryoprotectant such as 5–15% glycerol or dimethyl sulfoxide (DMSO) is added to the cell suspension. These agents lower the freezing point of the solution, reducing ice crystal formation and minimizing cellular damage. 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 once the temperature drops below -25°C, and then the cells are transferred directly into liquid nitrogen after reaching -80°C. For thawing, the frozen tubes are quickly placed in a 40°C water bath.
**Materials required:** Cryotubes, centrifuge tubes, cell culture flasks, 500 mL beakers, tape, enamel cups, and 75% alcohol-soaked cotton swabs.
**Reagents needed:** Cell culture medium (RPMI-1640 or DMEM), fetal bovine serum, 0.25% trypsin solution, DMSO or glycerol, 0.5% trypan blue, and liquid nitrogen.
**Equipment necessary:** A 4°C refrigerator, a -70°C freezer, a liquid nitrogen tank, a micropipette, a water bath, and a centrifuge.
**Procedure for cell cryopreservation:**
1. Detach the cells using trypsinization, then wash them with culture medium. Centrifuge at 800 rpm for 5 minutes, discard the supernatant, and collect the cell pellet.
2. Resuspend the cells in a cryopreservation solution containing 10% DMSO or glycerol mixed with 90% culture medium. Aim for a concentration of about 3 × 10ⶠcells/mL, and increase the serum concentration to 20% if possible.
3. Transfer the cell suspension into cryotubes, label them with details such as cell type, date, and freezing conditions, and wrap them with tape.
4. Freeze the tubes gradually: first at 4°C for 30 minutes, then at -20°C for 1.5 to 2 hours, followed by -70°C for 4–12 hours before transferring to liquid nitrogen. Keep a record of all steps.
**Procedure for cell resuscitation:**
1. Heat an enamel cup with tap water to around 40°C.
2. Remove the frozen tube from liquid nitrogen and quickly thaw it in the warm water.
3. Wipe the tube’s nozzle with 75% alcohol and open it carefully.
4. Centrifuge the cells, wash them once with serum-free medium, and resuspend in 3 mL of fresh culture medium. Seed the cells into a small culture flask.
5. Monitor the cells daily. If many dead cells are observed, change the medium the next day. Once the cells reach confluence, perform subculturing as needed.