In my role as global product manager of cold storage and liquid nitrogen cryopreservation for Thermo Fisher Scientific, I’m often asked which temperature is “best” for long-term cryopreservation.
My answer almost always starts the same way: “There’s no one-size-fits-all protocol for long-term storage at low temperatures.” Since each sample type has its own unique characteristics, each sample type will require its own cryopreservation process.
Of course, there are fundamental similarities across virtually all protocols. For example, when you’re designing a cryopreservation strategy:
You need to choose the right cryopreservation agent (CPA). Last month, I wrote about research suggesting that 2 or 3% glycerol may be optimal for boar sperm cryopreservation. But what works best for boar sperm could be detrimental to a different cell type or sperm from other animals. Review the literature to help determine which CPA is likely to be the most suitable for your samples.
You need to choose the right cooling rate. Over the years, it has become quite clear that optimal cooling rates can varywidely between sample types. For instance, embryonic stem cells do best when cooled at 0.5°C/min, while red blood cells should be preserved in the range of 200-1,000°C/min. Sub-optimal cooling rates can have a negative effect on sample viability once cells are thawed, so once again, make sure you consult the literature for recommendations before finalizing your protocol.
You need to choose the right storage temperature. By its very definition, cryopreservation is a way to preserve biological samples (tissue or cells) in a vitrified state for the purposes of long-term storage. (By “vitrified,” I mean a rapid version of freezing that prevents the formation of ice crystals by taking a sample to the amorphous ice phase, circumnavigating the hexagonal ice phase.) However, once frozen, different samples require different storage temperatures.
Let me share a few general guidelines. If your sample is:
- A cell component (DNA, RNA, etc.), it can typically be stored at -20°C, -40°C, -80°C.
- Bacteria, it can typically be stored at -80°C.
- Whole tissue (bone marrow, stem cells, sperm, etc.), finding the optimal storage temperature is more complex. If the cellular structure of a sample must remain intact, you’ll need to design a specific protocol to optimize viability. For whole cells from mammals, fish and plants, this usually involves temperatures of -130°C and colder.
(-130°C is the glass transition point of water and virtually all biological activity is stopped at these temperatures, allowing samples to be preserved for months or years without much damage.)
Please keep in mind that these are only guidelines. Remember to research the literature for recommendations before finalizing your cryopreservation protocol and long-term storage temperature.