Freezing Cells

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.

Freezing medium
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.

• Recovery Cell Culture Freezing Medium is a ready-to-use complete cryopreservation medium for mammalian cell cultures, containing an optimized ratio of fetal bovine serum to bovine serum for improved cell viability and cell recovery after thawing.
• Synth-a-Freeze Cryopreservation Medium is a chemically defined, protein-free, sterile cryopreservation medium containing 10% DMSO that is suitable for the cryopreservation of many stem and primary cell types with the exception of melanocytes.

Materials

• Culture vessels containing cultured cells in log-phase of growth
• Complete growth medium
• Cryoprotective agent such as DMSO (use a bottle set aside for cell culture; open only in a laminar flow hood) or a freezing medium such as Synth-a-Freeze Cryopreservation Medium or Recovery Cell Culture Freezing Medium
• Disposable, sterile 15-mL or 50-mL conical tubes
• Reagents and equipment to determine viable and total cell counts (e.g.,Invitrogen Countess II FL Automated Cell Counter, or hemocytometer, cell counter, and Trypan Blue)
• Sterile cryogenic storage vials (i.e., cryovials)
• Controlled rate freezing apparatus or isopropanol chamber
• Liquid nitrogen storage container

For freezing adherent cells, in addition to the above materials, you will need:

• Balanced salt solution such as Gibco Dulbecco’s Phosphate Buffered Saline (DPBS), containing no calcium, magnesium, or phenol red
• Dissociation reagent such as trypsin or Gibco TrypLE Express, without phenol red