Storage of Living Cells in Culture

These guidelines cover the low-temperature preservation and storage of living cells by freezing and freeze-drying. The cells covered by these practices include microorganisms (bacteria, fungi, protozoa, algae and viruses), mammalian tissue cultures, and plant cells.

Freezing of living cells is accomplished by suspending the cells in growth medium containing a cryoprotective chemical agent.

Freeze-drying (lyophilization) provides an alternative to storing living cells as frozen suspensions.

• Aluminum supports for freezer vials
• Dimethylsulfoxide (DMSO)
• Freezer vials
• Glass ampoules or plastic vials
• Glycerol
• Inventory system
• Liquid nitrogen freezer
• Mechanical freezer
• Programmable freezing unit
  
  
  

Methods

Freezing

1. Harvest cells from late logarithmic growth.
2. Suspend cells in fresh growth medium with 5%-10% dimethylsulfoxide (DMSO) or glycerol, and allow to equilibrate with the cryoprotectant for approximately 30 minutes prior to freezing. The cell suspension is dispensed in 0.5ml amounts into 1.0-1.2ml glass ampoules or plastic vials.
3. Freeze the suspensions by placing the ampoules or vials into a -60ºC freezer for 90 minutes, or cool at 1ºC per minute in a programmable freezing unit to -40ºC.
4. Following freezing, remove the ampoules or vials to a low-temperature freezer for storage.
5. Design inventory systems for frozen specimens to minimize handling during retrieval activities. This can be accomplished by using a cane storage system that allows retrieval of a single ampoule or vial from a liquid nitrogen freezer without exposing any other material to warmer temperatures (Fig. 1).
   
   
   
   Figure 1. Aluminum cane supports for freezer vials.
   
   
6. Constantly monitor mechanical freezers at -60ºC for temperature changes. Both visual surveillance, and if possible, surveillance by an automatic alarm system that can alert staff to potential temperature changes are recommended. Liquid nitrogen freezers should be set up to provide temperatures above the liquid of -150ºC or less when working in the unit. This requires maintaining, and monitoring an adequate liquid level, preferably by electronic surveillance connected to an alarm system.
7. Observe safety precautions when working with frozen material, especially in liquid nitrogen freezers. Cryogenic temperatures (<-100ºC) present extremely cold working environments and protective clothing should be worn. When working in the liquid portion of a liquid nitrogen freezer, all exposed skin should be protected, and a full face and neck shield should be worn to protect against splashing liquid and potentially exploding ampoules.
   
   Liquid nitrogen must always be handled in well-ventilated areas, and should never be placed into a sealed container. Nitrogen can quickly displace oxygen, especially during fill operations. Large facilities with more than three liquid nitrogen freezers should be equipped with an oxygen sensor to alert staff to low oxygen situations. All piping used to transport liquid nitrogen should be adequately equipped with safety relief valves to ensure against high pressure accidents.
   
   

Freeze-drying (lyophilization)

1. Harvest cells from late logarithmic growth.
2. Suspend in a medium suitable for freeze-drying such as skim milk, serum or various sugars. (DMSO and glycerol suspensions cannot be freeze-dried.)
3. Adequately freeze cells. Use slow, controlled cooling if necessary.
4. For long term storage, flame-seal glass ampoules, or use vials with butyl closures. After freeze-drying is complete, seal the containers in a vacuum, or in an inert gas atmosphere such as nitrogen. Freeze-dried cultures may be stored at 2-8ºC or lower, but do not require cryogenic temperatures.
5. Observe safety precautions during freeze-drying because of the potential for contamination of the freeze-drying apparatus from culture material that leaves the ampoule or vial during sublimation. Therefore, decontamination of the system is necessary unless bacteriological filters are used to protect the system during drying.
   
   When opening lyophils containing hazardous cultural material, take care not to aerosolize the vial contents, and perform all work with biohazards in a biological safety cabinet.
   
   

For long term stability, the frozen cells are maintained below -150ºC in a liquid nitrogen freezer. Some microorganisms can be maintained at -60ºC for several years. However, mammalian cells, plant cells, and protozoa and algae must be maintained below -150ºC for long term stability. These complex cells also require precise control of the cooling rate, and in some cases adjustments to the cooling protocol to overcome the detrimental effects of undercooling and liberation of the latent heat of fusion during freezing.

Maintenance of frozen specimens in the liquid portion of liquid nitrogen freezers is not recommended because of the potential for explosion of improperly sealed vials when material is retrieved.

It is advisable to maintain a small number of ampoules or vials of each culture at a separate physical location as safety material in the event of a physical disaster at the primary location.

To ensure that material preserved by freezing or freeze-drying remains unchanged, a program for characterization of the cells prior to, and following, preservation is a critical element in any collection of living cells.