Cryopreservation is a technique used to preserve living cells for extended periods by cooling them to extremely low temperatures, typically between -80°C and -196°C. This suspends their metabolic and biochemical activities. The purpose is to store valuable cell lines for future use without compromising viability or function, allowing researchers to maintain cultures, prevent genetic changes, and create backup cell banks.
The Science of Cell Preservation
Freezing cells presents biological and physical challenges that compromise viability. A primary threat is ice crystal formation, which causes mechanical damage by puncturing cell membranes or distorting structures. As water freezes, it excludes solutes, increasing salt concentration in unfrozen water, leading to osmotic shock and cellular dehydration. This “solution effect” also damages cells.
To mitigate these threats, cryoprotective agents (CPAs) like dimethyl sulfoxide (DMSO) and glycerol are used. CPAs penetrate cell membranes, increasing intracellular solute concentration and lowering water’s freezing point. This reduces large, damaging ice crystals and minimizes cellular dehydration. They also form hydrogen bonds with biological molecules, displacing water to help cells retain their native structure. However, CPA concentration and exposure time must be carefully managed to avoid toxicity.
Step-by-Step Cell Freezing
Freezing cells begins with preparation to ensure optimal post-thaw viability. Cells should be healthy, in logarithmic growth phase, and exhibit at least 90% viability. For adherent cells, gentle detachment from the culture vessel is necessary to minimize damage. Cells are counted to determine the appropriate density for freezing, often aiming for 1-2 million cells per milliliter.
A freezing medium containing cryoprotectants, such as 10% DMSO, is added to the cell suspension and aliquoted into sterile cryogenic vials. The cooling process is controlled to prevent rapid ice crystal formation. A slow rate, around -1°C per minute, is achieved using a controlled-rate freezer or an isopropanol-filled container (e.g., “Mr. Frosty”) placed in a -80°C freezer overnight. This gradual cooling allows cells to dehydrate partially without forming intracellular ice. After initial freezing, vials are transferred for long-term storage in the vapor phase of liquid nitrogen tanks, where temperatures are maintained below -130°C.
Reviving Frozen Cells
Reviving cryopreserved cells requires rapid thawing to minimize damage from ice recrystallization and prolonged cryoprotectant exposure. Vials are removed from liquid nitrogen storage and immediately placed into a 37°C water bath, with gentle agitation, until only a small ice crystal remains. This rapid warming, ideally completed within 1-2 minutes, is crucial for cell recovery.
After thawing, cells are transferred to a sterile tube containing pre-warmed growth medium. This dilutes and removes the cryoprotectant, which can be toxic if left in contact too long. A gentle centrifugation pellets the cells, allowing removal of the supernatant. The cell pellet is then resuspended in fresh growth medium and transferred to a culture vessel. Post-thaw cell viability is assessed using methods like visual inspection or trypan blue exclusion.