The field of cryopreservation has evolved over time in leaps and bounds. Freezing solutions have been refined and optimized, and new developments have enabled longer storage periods with better recovery rates. For the best results, below are 3 key tips that researchers should follow when freezing live cells or tissues.
Like many other great discoveries throughout history, the first cryopreservation media was made by accident. A team of researchers, Polge, Smith, and Parkes, worked on fowl spermatozoa back in 1948 and wanted to save some samples for later experiments. It is unsure how it happened, but instead of freezing the samples in a solution then used to preserve cells, they placed the cells in a mislabeled bottle containing a mixture of glycerol, albumen, and water. And, lo and behold, this new mixture worked far better than the intended solution, and thus the field of cryopreservation was born.
From those days of yore, the field has evolved and the solutions (hopefully not mislabeled) have been refined and adapted. It is now possible to store live cells and tissues at -20°C or -80°C, and new developments have enabled longer storage periods with better recovery rates. In addition, cryopreservation is also done at -196°C using liquid nitrogen (either in liquid or vapor form), which has become the staple for storing important material for long periods of time.
Top 3 Tips for Cryopreserving Cells
There are many different protocols for handling cryopreserved cells, each varying based on the intended use, time frame for preservation, availability of refrigerators and dewars, etc. Even so, all cryopreservation protocols have a few common points that researchers should follow for best results.
Use what’s best for your cells. Whether using “homebrew” or a commercial freezing medium, be sure to use what’s best for your cells or tissue. Sometimes optimization information can be found in relevant papers for specific cell types or applications. You can find the optimal media for your specific cells by screening multiple formulations. Assess each for cell recovery, attachment (if applicable), and proliferation after a thaw. Most cell freezing media are DMSO-based (in different percentages), but there are glycerol-based solutions as well (often used for bacteria). If you have sensitive cells, like stem cells, consider using a defined, animal component-free medium, such as BI’s NutriFreez™ D10 Cryopreservation Medium, to give you more control over the cells’ surroundings and greatly improve the freezing and thawing efficiency.
Pay attention to speed. When freezing samples, it is important to pay attention to your speed. Once freezing medium is added to the cells, work as fast as possible, since cells should have limited exposure to the DMSO in the freezing medium at room temperature. Of course remember to never sacrifice sterility and personal safety for speed! On the other hand, don’t chuck your vials straight in the liquid nitrogen or in a box in the fridge. Use a “Mr. Frosty” or similar freezing container that allows the samples to be cooled at a rate of 1°C per minute. Place the Mr. Frosty in the -80°C freezer overnight, and then transfer the vials to liquid nitrogen.
Choose the right storage location. Where to place your cryo vials? Inside the liquid nitrogen or in the vaporous layer? Some researchers claim that storage in the vaporous layer is better, stating that the temperature difference between the two phases is not great and/or that cells stored in the vapor phase won’t be exposed to potential mycoplasma contamination, which can be carried through the liquid nitrogen and is notoriously difficult to eliminate.
In conclusion, remember, safety is all-important, so handle your frozen vials with care, your freezing media with the appropriate biosafety measures, and take your time working carefully.