Freezing cells, or cryopreservation, is fundamental in biological research and biotechnology. This technique allows for the long-term storage of living cells while maintaining their viability and genetic integrity. Cryopreservation halts cellular metabolism by cooling cells to very low temperatures, typically between -80°C and -196°C. This state safeguards valuable cell lines for future experiments, ensuring consistency and reducing contamination or genetic changes from continuous culturing.
Preparing Cells for Freezing
Successful cell freezing requires careful preparation to maximize cell survival. Cells in their logarithmic growth phase generally yield the highest viability after thawing. Before freezing, it is important to confirm the cells are free from microbial contamination, with some protocols recommending mycoplasma testing.
Adherent cells must first be detached, often using an enzymatic solution like trypsin. Once harvested, cells are collected and concentrated by centrifugation. The cell pellet is then resuspended in a specialized freezing medium containing cryoprotective agents (CPAs), such as dimethyl sulfoxide (DMSO) or glycerol. These agents prevent damaging ice crystals inside cells during freezing. CPAs work by lowering the solution’s freezing point and promoting a glassy, non-crystalline water state, protecting cell membranes and organelles. A common DMSO concentration is 10% by volume.
The Freezing Process
Freezing cells requires a controlled-rate process to minimize cellular damage and ensure cell survival. Rapid cooling can lead to large, destructive ice crystals within cells. To counteract this, a gradual temperature decrease, typically around -1°C per minute, is recommended. This slow cooling rate allows water to move out of cells, concentrating intracellular solutes and reducing intracellular ice formation.
This controlled rate can be achieved using a programmable controlled-rate freezer. A more accessible method uses specialized freezing containers, often referred to as “Mr. Frosty.” These containers, filled with isopropyl alcohol and placed in a -80°C freezer, provide an insulated environment facilitating a cooling rate of approximately -1°C per minute. After several hours (usually overnight) in the -80°C freezer, cryovials are transferred to long-term storage. For long-term preservation, cells are stored below -135°C in liquid nitrogen freezers, either in liquid or vapor phase.
Thawing and Revival
Thawing frozen cells is as important as freezing for ensuring their viability and successful recovery. Unlike freezing, thawing should occur rapidly to prevent large, damaging ice crystals during rewarming. Cryovials should be quickly transferred from liquid nitrogen storage to a 37°C water bath. Swirl the vial gently until only a small ice crystal remains (usually within 1-2 minutes), indicating thawing. Avoid fully submerging the vial cap to prevent contamination.
Immediately after thawing, the cryoprotective agent must be diluted and removed to prevent its toxic effects. The thawed cell suspension is transferred dropwise into a sterile tube containing pre-warmed fresh culture medium. This dilutes the CPA concentration. For many cell types, a centrifugation step follows: cells are gently pelleted, and the supernatant containing the diluted CPA is discarded. The cell pellet is then resuspended in fresh, complete culture medium and transferred to an appropriate culture vessel. Cells are then placed in an incubator under suitable conditions for recovery and growth.
Maintaining Cell Health Post-Freezing
After thawing and initial plating, attention to cell health is important for recovery. Cells experience stress during the freeze-thaw process, and their initial recovery requires careful handling. Observing cells under a microscope for proper attachment (for adherent cells) and healthy growth is routine.
Assessing cell viability is common, often using methods like trypan blue exclusion, where only cells with compromised membranes take up the dye. While immediate post-thaw viability provides a snapshot, some cells may appear viable initially but undergo delayed damage or programmed cell death (apoptosis) hours or days later. Continuous monitoring for microbial contamination (e.g., media turbidity or changes in cell morphology) is important. Maintaining a clean and controlled environment ensures the long-term health and stability of the revived cell population.