Good Cryogenic Practices for Storing Biological Samples in ULT Freezers

Although many researchers routinely use ultra-low temperature (ULT) freezers, few of us are aware of all the steps we can take to truly protect our samples. In May 2012, a ULT freezer at the Harvard Brain Tissue Resource Center, a renowned brain bank, failed, damaging one-third of the world’s largest collection of autism brain samples. The external thermostat on the freezer read -80°C, but the temperature inside was actually that of a refrigerator. The loss of the samples critically set back autism research, underscoring the importance of implementing best practices for cryogenic preservation. In this edition of Bench Tips, we recommend four tips for safekeeping your samples in ULTs.

Build in redundancy

Most labs arm freezers with alarm systems but fail to put independent temperature monitors and adequate backup systems in place. The McLean freezer, one of 24 at the Harvard Brain Tissue Resource Center, was protected by two separate alarm systems. Staff members checked an external thermostat twice a day to ensure that the tissue samples were maintained at -80°C. Yet as the temperature rose, the external thermostat continued to read -79°C and therefore failed to trigger the alarms.

If the lab had had independent temperature monitors connected to the alarms, running through a separate port into the freezer, the samples might have been spared. Another good investment is a CO2 or liquid-nitrogen backup system. In the event of freezer failure, the system can release gas into the freezer, buying you extra time to transfer samples to a functional unit. Make sure there is always a backup freezer of similar capacity to hold transferred samples.

Protect against freezer burn

Freezing is a complex phenomenon that is still not fully understood. Various freezing temperatures create different kinds of ice crystals, which form at different rates. In a -80°C environment, stable hexagonal crystals are formed, but transitioning into and out of this stable frozen state is a delicate process. Generally the larger the cells the more critical it is to cool slowly. A cooling rate of 1°C per minute in ambient temperature is best, even with more tolerant permeable cells. Adding a cryoprotective additive beforehand—like DMSO or glycerol—can protect cells during freezing from increased solute concentration and ice-crystal formation. DMSO encourages greater dehydration of the cell prior to intracellular freezing, whereas glycerol is a less-toxic alternative. Both DMSO and glycerol are typically used in concentrations of 5% to 10% (v/v), but they’re rarely used together, with the exception of research with plant cells. Use the highest concentration the cell type is known to tolerate and sterilized, reagent-grade solutions. Conversely, thawing should occur quickly for most cells, using a 37°C water bath when possible.

Tailor conditions to cell type

Cell type, cell viability, growth conditions and physiological state of the cells are just some of the factors to evaluate before preparing cells for cryopreservation. In plant cells, it is optimum to harvest cells from the late-log phase and use a two-step cooling process—holding the cells at -30°C for a period of time before freezing at -80°C. Microbial cells, particularly bacteria and yeast grown under aerated conditions, demonstrate greater resistance to the detrimental effects of cooling and freezing than do nonaerated cells. When freezing fungal spores, make sure germination doesn’t occur prior to freezing. Determine viability and estimate recovery before and after freezing a culture via cell counting. A comparison of counts will indicate the success of your technique. In general, the greater the number of cells initially present, the greater the recovery.

Actively organize

It’s important that every lab establish a -80°C storage protocol. Develop a no-exception toss-it-out rule for all unmarked samples. Lab members should only get freezer access after they have cataloged their samples in a central location, which must also be backed up regularly. Pay special attention to labeling. Blue permanent markers tend to fade and become illegible in the freezer. Information written directly on ethanol-preserved samples almost inevitably wears off on polypropylene tubes. Many stick-on labels fall off in ultra-lows; test a few different types before committing to a large-scale purchase. Pay special attention to the gaskets on screw-ring tops, which can crack over time. An annual defrost and cleaning of all stored material keeps records straight and potentially spares the lab from purchasing another freezer for overflow. Also, remember that most researchers store at -80°C out of habit. Keep in mind that the lower you go in temperature, the more energy is consumed. Certain types of samples—microbial cultures, yeast and fungi—are fine in -70°C storage. As a final word of advice, signs of excess energy consumption—like condensation, water leaks in front of the units, overheating rooms and loud sounds—signal a freezer in trouble. Keep an eye out and perform regular maintenance; such steps should keep your freezers safe and your samples viable for years to come.

Co-Authored by Daniela Marino, Product Manager, Eppendorf

Related Products from: Eppendorf North America