Cryopreservation, the storage of biosamples at temperatures low enough to maintain biological integrity and prevent degradation, has its own specific set of safety requirements. In addition to employing good laboratory working practices with adequate training, associated staff and biobank managers need to be aware of the hazards associated with working with biological materials at extremely low temperatures.
Staff should guard against accidental skin exposure to low temperatures while preparing or accessing cryopreserved samples. This means wearing padded gauntlets for hand/arm protection, long-sleeved lab coats and, of course, closed-toe footwear.
Where operators are dealing with liquid nitrogen and exposed to temperatures well below the -20°C or -80°C of commercial freezer units, they should take care to avoid exposure to both the liquid and the vapor phase of this storage medium. This means working in areas with sufficient ventilation in addition to wearing full face/neck protection to protect vulnerable areas from splashback. In most cases, a full-face visor with neck guard is advisable.
Where walk-in freezer units are in use, training staff to comply with safety procedures in place for accessing these storage areas is important. Managers have a duty to ensure all alarm systems are properly functioning, and that 24-hour monitoring is in place in case of accidental entrapment. From personal experience, it is best not to locate these units in basements or areas of low traffic!
Preparing biological samples for cryopreservation frequently involves the use of additives as cryopreservatives. These include compounds such as dimethyl sulfoxide (DMSO), polyethylene glycol and glycerol, which protect the cells against damage during freezing. They also minimize risks associated with increased solute concentration and ice crystal formation. Staff should take special care when using DMSO; although it is non-toxic, it is easily taken up through skin exposure and can act as a carrier for more dangerous materials. When preparing samples with this reagent, biobank staff should wear long-sleeved lab coats, face protection and gloves to cover exposed skin.
Biohazards and cryopreserved samples
Dealing with biosamples requires attention to appropriate biosafety levels, including being aware of the potential for infection and setting in place procedures for known pathogens according to standard protocols. Alongside sound laboratory practice for preventing exposure to biohazards, there are several concerns specific to cryopreservation, such as storage contamination and potential for explosive sample dispersal.
Labeling is important in all forms of biobanking, and especially for biohazardous materials. Staff should know in advance what safety procedures they require for the samples they deal with, especially when working with human and primate cells. These include using appropriate personal protective gear and biosafety cabinets for containment.
Staff should also take precautions when retrieving samples from cryostorage. Samples cryopreserved in liquid nitrogen present a risk of explosion, especially if stored (deliberately or accidentally) in the liquid phase of this medium. This happens when liquid nitrogen enters a storage vial through the cap’s screw thread or via microchannels in an improperly heat-sealed glass vial. When the vials are brought to a room-temperature environment, the liquid nitrogen expands rapidly, with potentially explosive consequences. Precautions that staff should take include the following:
- Allow samples retrieved from the liquid phase to equilibrate for 24 hours in the vapor phase before thawing them.
- Store samples in the vapor phase to minimize liquid nitrogen entering the storage tube.
- If storing tubes are submerged, consider protecting them from contamination by wrapping them in CryoFlex to prevent contact with liquid nitrogen.
- Use appropriate biosafety cabinets when thawing biosamples retrieved from liquid nitrogen to contain the spread of materials if the vial explodes.
Staff also need to be aware that pathogens can survive the extreme low temperatures of liquid nitrogen storage; this includes survival in the liquid phase. It is important, therefore, to wipe the outside of vials with gauze soaked in alcohol before opening them to retrieve contents.
Where contamination is suspected, staff should follow appropriate decontamination procedures for cleaning liquid nitrogen storage tanks, minimizing exposure and preserving sample integrity.
A Guide for Proper Cryogenic Preservation (2012). Written by Frank P. Simione, M.S. of the American Type Culture Collection (ATCC), in cooperation with Thermo Fisher Scientific
- Thermo Scientific cryostorage webinar
- Thermo Scientific’s webpages on cold storage and cryopreservation
- A Guide to Cryopreservation Techniques (2004). Written for Thermo Scientific by Dr. Kelvin G. M. Brockbank, James C. Covault and Dr. Michael J. Taylor