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On cell cryopreservation, resuscitation procedures, and the applications of cryopreservation media.
Cell cryopreservation is a technique that involves storing cells at low temperatures to reduce cellular metabolism, enabling long-term preservation. This allows cells to be temporarily suspended from active growth while maintaining their biological characteristics, so they can be revived for experimental use when needed. Moreover, maintaining an appropriate stock of cells helps prevent loss due to contamination or other unforeseen events, thereby serving as a vital cell bank.
Cell Cryopreservation Procedure
1. Prepare a cryopreservation medium containing 10% DMSO or glycerol and 10–20% fetal bovine serum.
2. Harvest cells in the logarithmic growth phase, discard the old culture medium, and wash with PBS.
3. Remove the PBS, add an appropriate amount of trypsin (to cover the surface of the dish), and digest the monolayer‑grown cells to detach them.
4. Centrifuge at 1000 rpm for 5 min;
5. Remove the trypsin, add an appropriate volume of prepared cryopreservation medium, gently pipette to resuspend the cells evenly, count the cells, and adjust the final cell density in the cryopreservation medium to 5 × 10^6/mL–1 × 10^7/mL.
6. Aliquot the cells into cryovials, 1–1.5 ml per tube;
7. Label the cryovials with the cell line name, the date of cryopreservation, and the operator’s name.
8. Cryopreservation: The standard cryopreservation protocol involves a cooling rate of −1 to −2°C/min; when the temperature drops below −25°C, the cooling rate may be increased to −5 to −10°C/min; at −100°C, the sample can then be rapidly plunged into liquid nitrogen. Alternatively, cryovials containing cells may be placed in a −20°C freezer for 2 hours, followed by overnight storage at −70°C; the vials are then removed and transferred to a liquid nitrogen container.
Cell Revival Procedure
1. Remove the cryovial from the liquid nitrogen container, immerse it directly in 37°C warm water, and shake it periodically to facilitate rapid thawing.
2. Remove the cryovial from the 37°C water bath, open the cap, pipette out the cell suspension, transfer it to a centrifuge tube, and add at least tenfold the volume of culture medium; mix thoroughly.
3. Centrifuge at 1000 rpm for 5 minutes;
4. Discard the supernatant, resuspend the cells in culture medium containing 10% fetal bovine serum, count the cells, adjust the cell density, seed them into culture flasks, and incubate at 37°C in a static culture environment.
5. The next day, replace the culture medium once and continue culturing.
Applications of Cell Cryopreservation Solution
1. Serum-free cryopreservation solution is used for the storage of hematopoietic stem cells, mesenchymal stem cells, and other types of stem cells.
2. Serum-free cryopreservation solution is used for the storage of immune cells such as T lymphocytes and NK cells.
3. Serum-free cell cryopreservation solution is used for the storage of other types of mammalian cells.
Precautions for Cell Cryopreservation
1. Cell cultures from the proliferation phase up to the stage when a dense monolayer has formed can all be cryopreserved; however, cells in the logarithmic growth phase are preferred. It is advisable to replace the culture medium one day prior to cryopreservation.
2. When placing cryovials into or removing them from a liquid nitrogen container, take appropriate protective measures to prevent frostbite.
3. It is best to use freshly prepared culture medium for cryopreservation and thawing.
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