Cells in culture are very sensitive to microbial contamination, can often have a short life span, and may also be prone to genetic changes. Any one of these factors can quickly ruin a culture, forcing you to prepare a fresh batch of cells – a process that’s time-consuming and expensive.
You can avoid such a hassle by cryopreserving batches of cultured cells in freezing medium containing a cryoprotectant (e.g. DMSO or glycerol) that protects them from any damage caused during the freezing process. Most cell types can survive for years when they’re cryopreserved, but their viability upon thawing depends on how you go about storing them.
Best practices when cryopreserving cells in your lab
1) Before freezing your cells, you must first ensure they’re healthy, contaminant-free, and have a viability of >90%. Once you’ve harvested your cells to be preserved, count and suspend them in the freezing medium to begin the freezing process.
2) Frequent freeze-thaw cycles can produce ice crystals, causing problems like cell rupture, changes in buffer concentrations or growth factor degradation. You can minimize the need to repeatedly freeze and thaw cells by cryopreserving cells in single-use cryo vials.
3) As a general guide, cells should be cooled at a rate of approximately 1°C to 3°C per minute, until they reach around -80°C. After this, transfer your cells into the vapor phase of liquid nitrogen tanks, where they’ll be kept at the optimal temperature of -140°C to -196°C for long-term storage. This gradual cooling procedure helps to maintain cell integrity; options you can find to help control the freezing rate include using programmable cell-freezing equipment or a simple, cost-effective cooling container such as Thermo Scientific™ Mr. Frosty™.
4) Unlike freezing, the thawing of cells needs to be done very quickly to ensure maximum survival rate. To thaw your cells, incubate the vial containing the frozen cells in a 37°C water bath until only a tiny sliver of ice is visible. To avoid contamination upon thawing, always wipe the cryo vials with 70% ethanol before opening. Then quickly transfer the cells into pre-warmed growth medium and remove the cryoprotectant by centrifugation and re-suspension.
In addition to these tips, it’s important to maintain an accurate inventory to ensure you always know how many cells you have in storage, and be sure to be ready to prepare more when you’re running low. After thawing a vial, it’s always a good idea to make a note to update your inventory. A fully up-to-date cryopreservation inventory could save countless hours down the line, should the worst ever happen to your cultures!
Looking for additional cryopreservation resources?
Please visit our Cell Culture Community to access additional information designed to support your cryopreservation process, including the Cryopreservation Manual located under Protocols for cell culture applications.