The study of the stability of biological samples is often one of the hot topics in the daily life of all analytical researchers. It is also favored by the majority of researchers in the international Forum on biological analysis. Among them, three of the top topics to be discussed and voted are directly related to the stability of biological samples, indicating its importance. Ensuring the stability of samples in biological matrix is the basic premise of biological sample analysis.

Based on this, the national drug regulatory authorities all suggested reanalysis of the actual study samples. According to the In Vitro Diagnostic Reagent Registration Application Data Requirements and Approval Document Format issued by the Market Supervision Administration in 2021, the stability research of in vitro diagnostic reagents should submit at least 3 batches of real-time stability research data of declared products stored under actual storage conditions until the finished product validity period. Specify storage environment conditions (such as temperature, humidity and light) and expiration date [1]. The international basic requirements on the stability of in vitro diagnostic reagents are consistent with those in China. In 1991, the US FDA issued the guiding principle "Shelf Life of Medical Devices" [2], which provided the consideration factors and verification principles for stability research, but did not involve specific verification methods and test items. Clinical and Laboratory Standards Institute, CLSI published the EP25-A Evaluation of Stability of In Vitro Diagnostic Reagents in 2009, It provides guidance for the stability study of quantitative and qualitative in vitro diagnostic reagents [3].

In recent years, biobank has developed rapidly, with dozens of human samples stored. However, each sample bank does not adopt standard and unified cryopreservation technology and methods, which may cause changes in required sample information in the long-term storage process. Therefore, on the basis of exploring the cryopreservation mechanism, the key to ensure the long-term preservation quality of biological samples is to select appropriate cryopreservation agent, rising and cooling rate and other methods to treat the samples, and store them in a reasonable temperature environment.

This temperature is the crystallization temperature of water, which is easy to cause damage to the microstructure of cells and tissues. Generally, this temperature is not used to preserve tissues and cells. In addition, the stability of biomacromolecules in tissue samples may be significantly reduced due to the influence of various factors in cell tissues. Therefore, -60~0℃ is usually not used as the storage temperature in the sample library.

-80℃ is the commonly used sample storage temperature, which is also the temperature commonly used ultra-low temperature refrigerator can reach. This temperature is also commonly used to preserve the activity of biomacromolecules in samples due to the ease of operation, storage capacity and cost.

But how long this temperature can be maintained for different biomacromolecular activities is still under investigation. Studies have shown that DNA production, integrity and RNA production can be maintained for up to 7 years without significant changes at -80℃, but RNA integrity will start to decline after 5 years of frozen storage. Therefore, for long-term preservation of RNA activity, it is recommended to use lower temperatures or to extract RNA from a small part of the sample and store it synchronously with the rest of the sample. Proteins, lipids and other biomacromolecules in other samples can also be preserved at -80℃, but the duration is different, and the stability gradually decreases.

-150℃ is lower than the glass transformation temperature of water (-136℃), which is the temperature that can be reached by gaseous liquid nitrogen tank and cryogenic refrigerator. In this low temperature range, biological activities of samples are greatly reduced, which is the ideal temperature for preserving cell activity in samples. Cryogenic refrigerator is electric refrigeration without liquid nitrogen, and the stable temperature after filling samples is usually below -140℃. Compared with the use of gas liquid nitrogen tank, its advantage is that it does not need to add liquid nitrogen frequently, and it is easy to maintain. However, the cooling speed of electric refrigeration is lower than that of liquid nitrogen. Once the container is opened to take samples, it is easy to cause a large range of temperature fluctuations, and the temperature recovery time is relatively long. Therefore, it is more suitable for the situation of less opening to take samples. In addition, the electric refrigeration refrigerator must be powered. Liquid nitrogen equipment is recommended to provide backup storage in case of power failure.

-196℃ is the temperature of liquid nitrogen volatilization, only liquid nitrogen liquid phase preservation technology can reach this temperature. sample

Repeated freeze-thaw should be avoided when samples are stored in stable conditions. Repeated freezing-thawing of samples will accelerate the degradation of biomacromolecules and seriously affect the quality of samples. Before freezing the samples, it is better to pack them into small samples for storage, which can avoid unnecessary repetition.