Cryopreservation and Storage of Cell Lines

1.Cryopreservation of Cell Lines

The aim of cryopreservation is to enable stocks of cells to be stored to prevent the need to have all cell lines in culture at all times. It is invaluable when dealing with cells of limited life span. The other main advantages of cryopreservation are:

Reduced risk of microbial contamination

Reduced risk of cross contamination with other cell lines

Reduced risk of genetic drift and morphological changes

Work conducted using cells at a consistent passage number (refer to cell banking section below)

Reduced costs (consumables and staff time)

There has been a large amount of developmental work undertaken to ensure successful cryopreservation and resuscitation of a wide variety of cell lines of different cell types. The basic principle of successful cryopreservation is a slow freeze and quick thaw. Although the precise requirement may vary with different cell lines as a general guide cells should be cooled at a rate of –1oC to –3oC per minute and thawed quickly by incubation in a 37oC waterbath for 3-5 minutes. If this and the additional points given below are followed then most cell lines should be cryopreserved successfully.

Cultures should be healthy with a viability of >90% and no signs of microbial contamination.

Cultures should be in log phase of growth (this can be achieved by using pre-confluent cultures i.e. cultures that are below their maximum cell density and by changing the culture medium 24 hours before freezing).

A high concentration of serum/protein (>20%) should be used. In many cases serum is used at 90%.

Use a cryoprotectant such as dimethyl sulphoxide (DMSO Prod. No. D2650) or glycerol (Prod. No. G2025) to help protect the cells from rupture by the formation of ice crystals. The most commonly used cryoprotectant is DMSO at a final concentration of 10%, however, this is not appropriate for all cell lines e.g. HL60 (Prod. No. 98070106-1v1) where DMSO is used to induce differentiation. In such cases an alternative such as glycerol (Prod. No. G2025) should be used (refer to ECACC data sheet for details of the correct cryoprotectant). Sigma also offers ready-made cell freezing media containing DMSO (Prod. No. C6164), glycerol (Prod. No. C6039) and a serum-free formulation containing DMSO (Prod. No. C6295).

2.Ultra-low Temperature Storage of Cell Lines

Following controlled rate freezing in the presence of cryoprotectants, cell lines can be cryopreserved in a suspended state for indefinite periods provided a temperature of less than -135oC is maintained. Such ultra-low temperatures can only be attained by specialized electric freezers or more usually by immersion in liquid or vapor phase nitrogen. The advantages and disadvantages can be summarized as follows: