Cryovial Roulette by Greg Kwolek

Cryopreservation, the use of ultra-low temperatures to preserve cell lines and other critical biological materials, has become common practice in modern research.  The associated physical hazards of liquid nitrogen and other cryopreservatives, however, carry the risk of serious injury, infection, and specimen contamination in the event of an incident involving cryopreserved materials.  Cryogenic storage vials, specifically, have the potential to rupture and become projectiles, or to violently disperse their contents upon removal from liquid phase nitrogen storage.  Liquid nitrogen can leak into the vials during immersion and rapidly expand when removed from storage and warmed to room temperature.  With a liquid to gas expansion ratio of 1 to 700, the rapid evaporation of liquid nitrogen creates a pressure gradient far too great for the thin high-density polyethylene (HDPE) walls of cryogenic storage vials to contain.  Nalgene, Nunc, and Corning, among other popular brands of cryovials used at Columbia University, all warn against the storage of their products in liquid phase nitrogen for these very reasons. NitrogenLiquid

A recent laboratory incident highlights the research safety implications of cryopreservation.  A graduate student was struck in the face when a cryovial, recently removed from storage under liquid nitrogen, violently ruptured.  The shattered vial narrowly missed the affected student’s eye, impacting their cheek and potentially contaminating their skin with the cells preserved within the vial.  A similar vial ruptured in a separate incident, sounding as loud as a gunshot and leaving the affected student with a temporary ringing in the ears.  Any of the thousands of vials immersed in liquid nitrogen on campus today present these same hazards, regardless of their manufacturer, design, or size.

Cryopreservation in liquid phase nitrogen presents additional risks to researchers beyond personal injury.  Documented cases of bacterial and viral cross-contamination through liquid nitrogen from leaking containers have been published.  In one case, bone marrow harvested for transplantation became contaminated with Hepatitis B virus (HBV) present in the liquid nitrogen, causing a small outbreak in the patients receiving the treatment.  Just as alarming was the presence of the harvested patients’ DNA in the liquid nitrogen, affirming that contaminants can move both in and out of storage containers.

Storage temperatures must be consistently maintained below the glass transition temperature (Tg) of water to ensure successful long-term cryopreservation.  Liquid phase nitrogen is very effective at cooling samples to a frosty -196°C (60°C below Tg), the temperature at which biological activity, including the mechanisms responsible for cell death, ceases.  However, several safer alternatives to liquid phase nitrogen storage are available.

Mechanical and liquid nitrogen refrigerated vapor phase storage systems are available in a variety of temperature ranges, price points, and storage capacities.  Current generation mechanically refrigerated cryogenic freezers can maintain storage temperatures to -150°C uniformly throughout the storage chamber.  Specimens are thus maintained safely below Tg without the use of liquid nitrogen, protecting the specimens from cross-contamination and preventing the researcher from engaging in a game of cryovial roulette. 

More commonly, liquid nitrogen refrigerated vapor phase storage solutions are employed.  Cryogenic freezers or liquid nitrogen dewars are partially filled, relying on the slow evaporation of liquid to cool the storage chamber.  When liquid nitrogen levels are managed appropriately, the vapor within the storage chamber can be maintained below -150°C, safely preserving the specimens.
While not the preferred method for cryopreservation, some laboratories may still be faced with immersing vials in liquid phase nitrogen due to research or other operational constraints.  When it is necessary to use this storage method, a variety of safety precautions must be closely adhered to.  Despite improving vial designs, such as internal threading and silicon o-rings, liquid nitrogen may still leak into vials through the cap threads.  Preventing direct contact between the cap threads and liquid nitrogen by hermetically sealing the vials inside heat-shrink tubing is the manufacturer approved method for safe liquid phase nitrogen storage. 

To further enhance safety, vials should first be removed to vapor phase storage for 24 hours to allow trapped liquid nitrogen to slowly boil away, before being moved to a biological safety cabinet or other suitable enclosure for further thawing.  Anyone manipulating cryogenic material or equipment should be wearing a buttoned laboratory coat, safety glasses, a full-face shield, and insulated gloves for personal protection.

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