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.

1. Cultures should be healthy with a viability of >90% and no signs of microbial contamination.
2. 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).
3. A high concentration of serum/protein (>20%) should be used. In many cases serum is used at 90%.
4. 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).

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7.2   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:

Table 3. Comparison of ultra-low temperature storage methods for cell lines.

Storage in liquid phase nitrogen allows the lowest possible storage temperature to be maintained with absolute consistency, but requires the use of large volumes (depth) of liquid nitrogen and sealed glass ampules. Both of these requirements create potential hazards. There have also been documented cases of cross contamination by virus pathogens via the liquid nitrogen medium. For these reasons ultra-low temperature storage is most commonly in vapor phase nitrogen.

For vapor phase nitrogen storage, the ampules are positioned above a shallow reservoir of liquid nitrogen, the depth of which has to be carefully maintained. A vertical temperature gradient will exist through the vapor phase, the extremes of which will depend on the liquid levels maintained, the design of the vessel, and the frequency with which it is opened. Temperature variations in the upper regions of a vapor phase storage vessel can be extreme if regular maintenance is not carried out.

All liquid nitrogen storage vessels should include alarms that at least warn of low liquid nitrogen levels. This is particularly true of vapor phase storage systems. The bulk liquid nitrogen storage vessel should not be allowed to become less than half full before it is resupplied. This will ensure that at least one delivery can be missed without catastrophic consequences.

Inventory Control
All ultra-low temperature storage vessels will include a racking / inventory system designed to organize the contents for ease of location and retrieval. This should be supported by accurate record keeping and inventory control incorporating the following:

• Each ampule should be individually labeled, using “wrap around”, liquid nitrogen resistant labels with identity, lot number and date of freezing
• The location of each ampule should be recorded ideally on an electronic database or spreadsheet, but also on a paper storage plan
• There should be a control system to ensure that no ampule can be deposited or withdrawn without updating the records

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7.3   Safety Considerations

General safety issues
It is important that staffs are trained in the use of liquid nitrogen and associated equipment including the storage vessels, which need to be vented safely, and containers, which may need to be filled. As with all laboratory procedures personal protective equipment should be worn at all times whilst handling nitrogen, including a full-face visor and thermally insulated gloves in addition to a laboratory coat. Proper training and the use of protective equipment will minimize the risk of frostbite and other minor incidents.

Risk of asphyxiation
The single most important safety consideration is the potential risk of asphyxiation due to the high levels of nitrogen that can lead to oxygen depletion. This is critical since oxygen depletion can very rapidly cause loss of consciousness, without warning.

Consequently liquid nitrogen refrigerators should be placed in well-ventilated areas in order to minimize this risk. Large volume stores should have low oxygen alarm systems.

Preventative measures

• Use oxygen alarms set to 18% oxygen (v/v)
• Staff training – staff should be trained to evacuate the area immediately on hearing the alarm and not return until the oxygen is back to normal (~ 20% v/v)
• Staff should work in pairs when handling liquid nitrogen
• Prohibit the use of nitrogen outside of normal working hours
• Mechanical ventilation systems should be installed if at all possible

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