There are a number of reasons for the storage and preservation of tissue culture cell lines. Cell lines subject to continuous culture are prone to selective variation, senescence, as well as genetic and phenotypic drift. Despite the best practices, equipment failure and contamination can ruin culture stocks. Additionally the storage rather than the maintenance of unneeded cell lines can be savings of time and materials. For these reasons and more, cultured cells are typically stored “frozen” at very low temperatures for extended periods of time.
Optimal freezing of cells for maximal viable recovery upon thawing minimizes intracellular ice crystal formation. This is accomplished through the use of cryoprotectants such as DMSO or glycerol. Freezing cells at a high cell-concentration allows for sufficient dilution of the cryoprotectant upon thawing and reseeding, making centrifugation to remove the cryoprotectant unnecessary. It has been reported that optimal cell survival occurs if cells are cooled at 1°C/min . This cooling rate is a compromise between fast freezing, which minimizes ice crystal formation and slow cooling, which encourages the extra-cellular migration of water.
Low temperature cell storage has a number of different options, each with advantages and disadvantages. The least expensive would simply be storage in a conventional -80°C electric freezer. Almost every cell biology laboratory has this type of freezer, making their use very appealing. Unfortunately the relatively high temperature of these devices, along with the continual opening of the access door to retrieve materials not slated for long-term storage makes these freezers less than ideal for long term viability. Loss of viability at this temperature has been estimated to be 5-10% per annum . Dedicated ultra-low -135°C electric freezers provide a better option in terms of viability. These dedicated freezers do not “suffer” from being opened continuously. However they are mechanically complex, expensive to purchase and require some sort of liquid nitrogen back-up. As with any electric device, interruptions in power can be catastrophic without some form of generator or battery backup.
For most users, long term cryopreservation of cell stocks employs the use of liquid nitrogen storage. In regards to liquid nitrogen storage one can use a liquid phase or a vapor stage storage device. With vapor stage nitrogen storage vials are positioned above a shallow reservoir of liquid nitrogen, the depth of which needs to be carefully maintained. The advent of improved insulation and reduced evaporation, along with automated monitoring of nitrogen levels, has made this the preferred method of liquid nitrogen storage. In this type of storage a vertical temperature gradient exists in the vapor from -190°C upward. Depending on the containment vessel and the frequency at which it is opened the temperature gradient will vary. Because of the small amount of nitrogen used in this system close monitoring of the liquid level is essential. Liquid phase storage submerges the vials directly in the liquid nitrogen. This method, while requiring more nitrogen, provides a constant temperature of -190°C. The larger capacity of fluid in the system means nitrogen will last significantly longer than a vapor system. Because the cells are immersed in liquid, the potential for cross-contamination of samples, while remote does exist and has been reported. One significant danger comes from liquid nitrogen that has leaked into cryovials. It has the potential to become explosive when it is thawed.
With either nitrogen storage system there are several different freezer designs to choose from based on their opening or neck size and the storage system employed. The most popular are the narrow neck designs, which reduce the rate of evaporative loss of nitrogen, but make access more difficult. These types typically store vials in either a cane that is placed in round canisters or in a series of trays that hold storage boxes. In either case the canisters or trays are attached to a long handle that hangs from the top lip of the storage vessel. Wide mouth designs have a similar arrangement, but allow easier access at the expense of higher evaporation rates. Automated systems are also available where nitrogen liquid level is monitored and added from an external reservoir automatically.
On the whole, long term storage of cell lines is an expensive yet an absolutely necessary endeavor for those investigators running cell based assays. Liquid nitrogen is non-flammable, provides an ideal storage temperature, readily available from a number of sources, and relatively inexpensive. Care should be taken when using liquid nitrogen. Asphyxiation is a real hazard if Liquid N2 is being replenished in a closed area. Additionally liquid N2 is extremely cold, care should be taken to avoid frostbite.
What type of long term cell storage is used in your lab?
1. Leibo, S.P. and P. Mazur, (1971) The role of cooling rates in low-temperature preservation. Cryobiology, 8:447-452.
2. Green, A.E., B. Athreya, H.B. Lehr, and L.L. Coriell (1967) Viability of Cell Cultures Following Extended Preservation in Liquid Nitrogen. Proc. Soc. Exp. Biol. Med. 124:1302-1307.