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.

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.

• 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

The following protocol describes a general procedure for cryopreserving cultured cells.  For detailed protocols, always refer to the cell-specific product insert.

1. Prepare freezing medium and store at 2° to 8°C until use. Note that the appropriate freezing medium depends on the cell line.
2. For adherent cells, gently detach cells from the tissue culture vessel following the procedure used during the subculture. Resuspend the cells in complete medium required for that cell type.
3. Determine the total number of cells and percent viability using a hemocytometer, cell counter, and Trypan Blue exclusion, or the Countess Automated Cell Counter. According to the desired viable cell density, calculate the required volume of freezing medium.

4. 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.

5. Resuspend the cell pellet in cold freezing medium at the recommended viable cell density for the specific cell type.
6. Dispense aliquots of the cell suspension into cryogenic storage vials.  As you aliquot them, frequently and gently mix the cells to maintain a homogeneous cell suspension.
7. Freeze the cells in a controlled rate freezing apparatus, decreasing the temperature approximately 1°C per minute. Alternatively, place the cryovials containing the cells in an isopropanol chamber and store them at –80°C overnight.
8. Transfer frozen cells to liquid nitrogen, and store them in the gas phase above the liquid nitrogen.

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.

• Freeze your cultured cell samples at a high concentration and at as low a passage number as possible.  Make sure that the cells are at least 90% viable before freezing. Note that the optimal freezing conditions depend on the cell line in use.
• Freeze the cells slowly by reducing the temperature at approximately 1°C per minute using a controlled rate cryo-freezer or a cryo-freezing container such as “Mr. Frosty,” available from Thermo Scientific Nalgene labware (Nalge Nunc).
• Always use the recommended freezing medium.  The freezing medium should contain a cryoprotective agent such as DMSO or glycerol (see What is Subculture?).
• Cell samples should be stored in vapor phase liquid nitrogen below –135°C.
• Always use sterile cryovials for storing frozen cells.  Cryovials containing the frozen cells may be stored immersed in liquid nitrogen or in the gas phase above the liquid nitrogen (see Safety Note below).
• Always wear personal protective equipment.
• All solutions and equipment that come in contact with the cells must be sterile.  Always use proper sterile technique and work in a laminar flow hood.