Cell lines in continuous culture are likely to suffer undesirable outcomes such as genetic drift, senescence, and microbial contamination, and even the best-run laboratories can experience equipment failure. An established cell line is a valuable resource, and its replacement is expensive and time consuming. Therefore, it is vitally important that they are frozen down and preserved for long-term storage. A properly maintained frozen cell stock is an important part of cell culture.
As soon as a small surplus of cells becomes available from subculturing, the best preservative method is to keep them frozen as a seed stock, protected, and not be made available for general laboratory use. Working stocks can be prepared and replenished from frozen seed stocks. If the seed stocks become depleted, cryopreserved working stocks can then serve as a source for preparing a fresh seed stock with a minimum increase in generation number from the initial freezing.
The general freezing method is the same for adherent and suspension cells, except that adherent cells need to be removed from the culture plates before starting the freezing procedure. The best method for cryopreserving cultured cells is storing them in liquid nitrogen in complete medium in the presence of a cryoprotective agent such as dimethyl sulfoxide (DMSO). Cryoprotective agents reduce the freezing point of the medium and allow a slower cooling rate, greatly reducing the risk of ice crystal formation, which can damage cells and cause cell death.
Note: A DMSO solution is known to facilitate the entry process of organic molecules into tissues. Handle reagents containing DMSO using equipment and practices appropriate for the hazards posed by such materials. Dispose of the reagents in compliance with local regulations.
Always use the recommended freezing medium for cryopreserving your cells. The freezing medium should contain a cryoprotective agent such as DMSO or glycerol. You may also use a specially formulated complete cryopreservation medium such as Gibco Recovery Cell Culture Freezing Medium or Gibco Synth-a-Freeze Cryopreservation Medium.
For freezing adherent cells, in addition to the above materials, you will need:
This video demonstrates the critical steps required to freeze cells while maintaining optimal cell health. We review the equipment required, how to prepare for freezing, and each step performed in a careful way at the right pace to prevent damage to your cells.
The following protocol describes a general procedure for cryopreserving cultured cells. For detailed protocols, always refer to the cell-specific product insert.
Centrifuge the cell suspension at approximately 100–200 × g for 5 to 10 minutes. Aseptically decant supernatant without disturbing the cell pellet.
Note: Centrifugation speed and duration varies depending on the cell type.
Following the guidelines below is essential for cryopreserving your cell lines for future use. As with other cell culture procedures, we recommend that you closely follow the instructions provided with your cell line for best results.
Safety Note: Biohazardous materials must be stored in the gas phase above the liquid nitrogen. Storing the sealed cryovials in the gas phase eliminates the risk of explosion. If you are using liquid-phase storage, be aware of the explosion hazard with both glass and plastic cryovials and always wear a face shield or goggles.
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