Vaccines, drugs, antibodies, enzymes, cells, blood, and scores of other biological materials are highly temperature sensitive. Fluctuations in temperature can reduce the efficacy, decompose, or shorten the shelf life of biologics. Therefore, it is important to store biologics at the right temperature using standardized protocols. Freezing in standard laboratory freezers set at -20 °C or -80 °C is the most popular mode of biological preservation. However, some biologics, including enzymes, may lose their efficacy at low temperatures, particularly if subjected to multiple cycles of freezing and thawing. For commercially available biologics, it’s best to follow manufacturer’s protocols when storing for long or short term.1
What, when, and why to freeze at -20 °C
Freezing at -20°C is ideal for biologics that are unstable at room temperature (20 °C) or in refrigerators (4 °C). Nucleic acids extracted from tissue samples are optimally dissolved in a stabilizing solution or lyophilized before storing at -20 °C. Freezing damages cell membranes and compromises histological integrity, therefore, biological specimens intended for histological or immunohistochemical analyses are ideally fixed and embedded before freezing. Since constancy of storage temperature is critical in maintaining the integrity of biological samples, it is useful to fit freezers with audio and visual temperature alarms, preferably those that alert responsible personnel elsewhere. Also, since most -20 °C are accessed frequently, it is best not to store samples on the door shelves because opening the freezer doors causes immediate exposure to ambient temperatures.
What, when, and why to freeze at -80 °C
For long-term storage of nucleic acids, tissue samples, or cultured cells, particularly those not suspended in stabilizing solutions, freezing at -80 °C is preferred. Freezing at -80 °C prevents the breakdown of nucleic acids, proteins, peptides, and other biomolecules. The viability of numerous biological assays, dependent on temperature-sensitive reagents including enzymes or antibodies, is best preserved at -80 °C. When preserving cultured cells at -80 °C, it is important to be mindful of the speed of freezing and thawing and the cell type involved. Live cells are best preserved when frozen slowly (about 1 °C per minute) but thawed rapidly (e.g., dipping in a water bath set to 37 °C). This prevents water crystallization that destroys cell membranes. Insulating screw-cap tubes before transferring them to the freezer slows down freezing of the contents and should be avoided. Storage at -80 °C is ideal for bacterial and yeast cells but not for mammalian cells. The most commonly used preservative for freezing live cells is glycerol. Bacteria are typically frozen in 15% glycerol at -80 °C for long-term storage. When retrieving bacteria, it’s sufficient to remove a tiny fraction of the frozen sample with the tip of a sterile pipette and streaking it on an agar plate. This obviates the need for thawing the entire sample, which can be quickly returned to storage.
Freezing samples without the use of liquid nitrogen can be accomplished in cryogenic freezers set to temperatures between -140 and -150 °C and is ideal for the long-term storage of biological samples. Samples preserved cryogenically are generally suspended in a stabilizing medium including a cryoprotectant. Mixtures of cryoprotectants such as formamide and DMSO have lower toxicity and greater efficacy than single-agent cryoprotectants.
Storage in liquid nitrogen (-196 °C liquid-phase temperature) is the gold standard in long-term freezing solutions. Freezing samples by immersion in a liquid maximizes the contact surface ensuring a uniformity of freezing temperatures, and the inert nature of nitrogen prevents chemical alteration of the specimen. Freezing at such low temperatures essentially suspends all biological activity and inhibits biological sample degradation due to nucleases and proteases. However, the disadvantage of liquid nitrogen is its low specific heat constant that causes it to boil when in contact with warmer samples. The resultant vapor barrier leads to differential temperatures on the surface and interior of the sample, leading to cracks in larger tissue samples.
In order to optimally freeze and store irreplaceable biological samples, it is important to consider the biochemical composition of the sample, the biomolecular structure of sample components, the use of preservatives or solutions in which the sample is dissolved or suspended, and the degree of sample integrity required for future applications. For large-scale and long-term freezing requirements, partnering with professional biostorage companies or biological repositories can be a viable option, particularly when lab space is at a premium.
- M.J. Redrup, et al., "Sample Management: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Team," AAPS J 18: 290-293, 2016.
- L. Gille, et al., "Effect of freezer storage time and thawing method on the recovery of Mycoplasma bovis from bovine colostrum," J Dairy Sci 101: 609-613, 2018.
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