Storage temperatures and monitoring are critical to meeting requirements for biobank organizations. While each project varies in requirements, it is essential to understand the benefits and risks associated with each temperature option. Investigators planning to use our biorepository often ask for advice on the optimal storage temperature for their samples. Based on my experience, I have found that there are 4 questions every investigator should ask to improve viability for long term storage.
- What is the type of sample? Are the samples biofluids (blood, serum, plasma, saliva, urine, or semen), whole cells, or tissues? Are they extracted components of cells nucleic acids, proteins, hormones) or complex mixtures of environmental material such as air, water, dust, or soil?
- What container is used for the sample? Are the containers made of glass or plastic that breaks easily at freezing temperatures, or of will they tolerate cryogenic temperatures?
- Which particular assays are planned? Will there be an analysis of specific components of the sample, like nucleic acids or proteins, or will enzyme activity or cell viability be the target measure?
- What length of time does the sample need to be stored? Will the samples be used in the short term, such as a week or month, or will they be stored for years?
As a rule, samples stored at temperatures colder than room temperature are better preserved. Cold inhibits destructive chemical reactions such as oxidation as well as degradation caused by enzyme activity (proteases, nucleases, lipases, etc.). Cold also inhibits the growth of any contaminating bacteria and molds. In general, the colder the better for long term storage.
However, freezing some kinds of samples can destroy their activity. For instance, proteins can undergo an irreversible structural change (denaturation) when frozen. Enzymes and antibodies can also lose much of their activity after freezing and thawing. If the end use of the sample is an assay for enzyme or antibody activity, then the sample usually can’t be frozen. Keep such samples refrigerated at +2 to +8⁰C. Pilot studies can determine how long the factor remains active under refrigeration; in general the sample may keep about one week. Some enzymes and antibodies can withstand a few cycles of freezing and thawing, and preliminary research should be done to determine if this is the case for the assays of interest. There are also commercially available cryoprotectants that allow antibodies to be stored frozen. Storing samples in small single-use aliquots avoids the need for more than one freeze/thaw.
Another type of sample that cannot be simply frozen is a living animal cell. To stay viable, a cryoprotectant such as dimethyl sulfoxide (DMSO) or glycerol must be added to the cells before freezing. Ideally, the cells should be frozen at a controlled rate (inexpensive devices exist for this purpose) to avoid temperature shock. Optimal conditions vary for the cell type being frozen, and preliminary experiments should be done if there are no references available in the literature. Tissues and organs should be treated similarly to whole cells, depending upon end use requirements. Formalin-fixed, paraffin-embedded (FFPE) tissue samples, either as paraffin blocks or cut and placed onto glass slides, can be stored at ambient temperatures (+15 to +30⁰C).
If all that is needed from the sample is extraction of nucleic acids for polymerase chain reaction (PCR) or genotyping, it is safe to freeze the sample in small aliquots for very long term storage. An ultralow (between -60 and -90⁰C) freezer is acceptable, or for even longer storage, they can be kept at -150 to -196⁰C in the vapor phase of a liquid nitrogen (LN2) freezer. DNA samples, whether purified or in the form of a spot of whole blood, can also be stored on commercially available filter paper at ambient temperatures, but for a more limited number of years, and humidity must be controlled.
Proteins needed for composition studies, or which survive freeze/thawing, can be stored in frozen aliquots and even lyophilized. Small aliquots avoid multiple freeze/thaw events. The aliquots can be stored in an ultralow freezer, or longer in an LN2 freezer. In addition, there is specially formulated filter paper commercially available for storing proteins at ambient temperatures, under controlled humidity.
Other sample components, either purified or still in the biofluid solution, including hormones, cytokines, vitamins, and minerals, can be stored in aliquots in either an ultralow freezer or an LN2 freezer, depending on the length of time storage is desired.
Storing bacteria is covered in another blog from Thermo Fisher Scientific on "Storing Bacterial Samples for Optimal Viability".
For some studies, environmental samples are collected and sent to the biorepository for long term storage. These can include samples of air, dust, water, and soil. Short term storage, a matter of months, can be done in a -20⁰C laboratory freezer (-10 to -25⁰C). Long term storage should be done in an ultralow freezer.
The sample containers are of concern if they are glass or if they are made of a material that becomes brittle and breaks easily at low temperatures. Cryogenic plastic containers should be chosen for samples in long-term frozen storage, when possible.
There are so many variables of biospecimen type, analyte of interest, and end use that is difficult to put a comprehensive table or diagram together. The purpose of this blog is not to dictate the storage temperature for these samples but to serve as a reference guide as well as emphasizing the notion that one must consider the questions in light of the science being accomplished. To clarify the complexity of various factors involved in biospecimen storage temperature, we have designed an illustrative poster.