Cryopreservation

Most cell cultures can be stored for many years, if not indefinitely, at temperatures below -130°C (cryopreservation). ATCC has recovered cells from cultures cryopreserved for more than 40 years. The many advantages of cryopreservation far outweigh the required investment in equipment and reagents. These advantages include:

• Generation of safety stocks to ensure against loss of the culture from equipment failures or contamination by microorganisms or other cell lines. 
• Elimination of the time, energy, and materials required to maintain cultures not in immediate use.
• Preservation of cells with finite population doublings (that will ultimately senesce).
• Insurance against phenotypic drift in the culture due to genetic instability and/or selective pressure.
• Creating a standard reagent to be used for a series of experiments.

As the cell suspension is cooled below the freezing point, ice crystals form and the concentration of the solutes in the suspension increases. Intracellular ice can be minimized if water within the cell is allowed to escape by osmosis during the cooling process. A slow cooling rate, generally -1°C per minute, facilitates this process. However, as the cells lose water, they shrink in size and will quickly lose viability if they go beyond a minimum volume. The addition of cryoprotectant agents such as glycerol or dimethylsulfoxide (DMSO) will mitigate these effects.  

The standard procedure for cryopreservation is to freeze cells slowly until they reach a temperature below -70°C in medium that includes a cryoprotectant. Vials are transferred to a liquid-nitrogen freezer to maintain them at temperatures below -130°C. 

The recovery of cryopreserved cells is straightforward: Cells are thawed rapidly in a water bath at 37°C, removed from the freeze-medium by gentle centrifugation and/or diluted with growth medium, and seeded in a culture vessel in complete growth medium. 

There are numerous factors which affect the viability of recovered cells. Modify the procedure for each cell line to attain optimal cell viability upon recovery. Some of the critical parameters for optimization include the composition of the freeze medium, the growth phase of the culture, the stage of the cell in the cell cycle, and the number and concentration of cells within the freezing solution. 

ATCC provides information on cryopreservation for all cell lines on the Product Sheet. Most ATCC cell lines are frozen with a cryopreservation medium consisting of 5% DMSO and complete growth medium.

Glycerol and DMSO at 5% to 10% are the most common cryoprotectant agents. While DMSO can be toxic to cells, it penetrates them much faster than glycerol and yields more reproducible results. Unfortunately, DMSO can cause some cells to differentiate (e.g., HL-60 promyeloblast cells) and may be too toxic for some cells (e.g., HBE4-E6/E7 lung epithelial cells). Glycerol should be used in these instances. Glycerol can be sterilized by autoclaving whereas DMSO must be sterilized by filtration. Care should be used when handling any DMSO solution as it will rapidly penetrate intact skin and may carry toxic contaminants along with it. 

Use only reagent-grade (or better, such as cell culture-grade) DMSO or glycerol. Store both in aliquots protected from light. ATCC offers DMSO (ATCC^® 4-X™) that has been thoroughly tested for cell culture use.

For cells grown in serum-free medium, adding 50% conditioned medium (serum-free medium in which the cells were grown for 24 hours) to both the cell freezing and the recovery medium may improve recovery and survival. The addition of 10% to 20% cell culture-grade bovine serum albumin to serum-free freezing medium may also increase post-freeze survival.

Serum-free freezing media have also been developed. ATCC^® Serum-Free Cell Freezing Medium (ATCC^® 30-2600™) can be used for both cells cultured in serum-supplemented growth medium as well as cells grown under serum-free conditions. This proprietary formulation contains 10% DMSO and methylcellulose is suitable for the cryopreservation of adherent and suspension cell cultures. Cells cryopreserved using Serum-Free Freezing Medium show levels of viability and percent attachment (adherent cells) that are comparable to cells preserved in DMSO and FBS. (See: Figure 3)

Figure 3. Cells cryopreserved using ATCC^® Serum-Free Cell Freezing Medium show levels of viability and percent attachment that are comparable to cells preserved in DMSO and FBS.

Other variations of freeze medium formulations include high (up to 90%) concentrations of serum which presumably supplies some cryoprotection as well as additional growth factors; use of a balanced salt solution designed for hypothermal conditions in place of medium designed for 37°C incubation; and the addition of apoptotic inhibitors which may prevent delayed onset cell death following recovery.²¹ Optimum formulations for individual cell lines need to be determined empirically.

Cryopreservation vials 

There are two materials to choose from for cryopreservation vials: glass or plastic. Glass vials are more difficult to work with; they need to be sterilized before use, they do not come with labels (information is imprinted into the glass), they need to be sealed with a hot flame, and they can be difficult to open. However, they are preferred for long-term storage (many years) of valuable cultures and are considered fail-safe once properly sealed. ATCC uses glass vials for the storage of seed stocks which are placed in the lower level of the liquid nitrogen tank. 

Plastic vials are used for the storage of distribution stocks. Plastic vials come in two varieties: those with an internal thread and silicone gasket and those with an external thread. The internal-thread version was the first commercially available, but has some disadvantages over the external-thread version. For example, while the silicone gasket provides an excellent seal, it needs to be tightened just right; too tight or too loose and the vial will leak.

Controlled-rate freezing chambers

There are several means to achieve a cooling rate of -1°C per minute. The best is with a computer controlled, programmable electronic freezing unit (such as CryoMed Freeze) which rigorously maintains this rate of cooling. This is the method used exclusively at ATCC. Such equipment is relatively expensive and absolutely necessary for only the most sensitive cells. A less costly approach is to place the cryopreservation vials into an insulated chamber and cool for 24 hours in a mechanical freezer at -70°C or lower. There are several commercially available freezing chambers which achieve a cooling rate very close to the ideal -1°C per minute (Mr. Frosty, Nalgene^® No. 5100-0001; or StrataCooler^®, Agilent Technologies No. 401349). Alternately, the vials can be placed into a polystyrene box with 15-mm (3/4 inch) thick walls and 1 L capacity packed with paper, cotton wool, or foam peanuts for insulation.

The procedure below will work for most cell cultures and should be modified as needed. Freeze medium formulations for all ATCC cell lines are provided on the Product Sheet. Harvest cells in exponential growth. 

1. Check your cell culture for contamination from bacteria, fungi, mycoplasma, and viruses (see section on Contamination) immediately before cryopreservation. In most cases, the results of the contamination screen will be available some time after the cultures are cryopreserved (10 to 14 days). If contamination is confirmed, then destroy the frozen material.
2. Prepare a freeze medium consisting of complete growth medium and 5% DMSO (ATCC^® 4-X™). Do not add undiluted DMSO to a cell suspension as dissolution of DMSO in aqueous solutions gives off heat.
3. Collect cells by gentle centrifugation (10 minutes at 125 × g) and resuspend them in the freeze medium at a concentration of 1 × 10⁶ to 5 × 10⁶ viable cells/mL. Continue to maintain the cells in culture until the viability of the recovered cells is confirmed (see Step 9).
4. Label the appropriate number of vials with the name of the cell line and the date. Then add 1 to 1.8 mL of the cell suspension to each of the vials (depending upon the volume of the vial) and seal. 
5. Allow cells to equilibrate in the freeze medium at room temperature for a minimum of 15 minutes but no longer than 40. This time is usually taken up in dispensing aliquots of the cell suspension into the vials. After 40 minutes, cell viability may decline due to the DMSO.
6. Place the vials into a pre-cooled (4°C), controlled-rate freeze chamber and place the chamber in a mechanical freezer at -70°C (or colder) for at least 24 hours. Alternately, use a pre-cooled (4°C) programmable freezer unit set to cool the vials at -1°C per minute until a temperature below -40°C is achieved and then set to abruptly drop to -130°C.
7. Quickly transfer the vials to a liquid nitrogen or -130°C freezer. Frozen material will warm up at a rate of 10°C per minute and cells will deteriorate rapidly if warmed above -50°C. 
8. Record the location and details of the freeze.
9. After 24 hours at -130°C, remove one vial, restore the cells in culture medium, and determine their viability and sterility.

The cell solution in the frozen vial needs to be warmed as rapidly as possible and then immediately combined with complete culture medium and seeded into an appropriate flask. While cells grown in monolayers can be recovered from cryopreservation in multiwell plates, the results are not as consistent as with flasks. 

Some cell lines, such as hybridoma cultures, take several days before they fully recover from cryopreservation. Some hybridomas show low viability on the first day in culture and will generate cellular debris. Viability for most cells declines and reaches a nadir at 24 hours post-thaw. Most, if not all, of this decline appears to be due to apoptosis (as opposed to necrosis) induced by the stress of the cryopreservation process.²² After this time point, cells begin to recover and enter exponential growth.

1. Prepare a culture vessel (T-75 flask) so that it contains at least 10 mL of the appropriate culture medium equilibrated for temperature and pH.
2. Remove the vial from the liquid nitrogen freezer and thaw by gentle agitation in a 37°C water bath (or a bath set at the normal growth temperature for that cell line). Thaw rapidly until ice crystals have melted (approximately 2 minutes).
3. Remove the vial from the water bath and decontaminate it by dipping in or spraying with 70% ethanol. Follow strict aseptic conditions in a laminar flow tissue culture hood for all further manipulations.
4. Unscrew the top of the vial and transfer the contents to a sterile centrifuge tube containing 9 mL complete growth medium. Remove the cryoprotectant agent by gentle centrifugation (10 minutes at 125 × g). Discard the supernatant, taking care not to disturb the soft pellet, and resuspend the cells in 1 mL or 2 mL of complete growth medium. Pipette gently to loosen the pellet and break apart clumps. (If the cells normally grow as clusters, avoid over-pipetting during resuspension.) Transfer the cell suspension into the medium in the culture vessel and mix thoroughly.
5. Examine the cultures after 24 hours and subculture as needed.