Cell cryopreservation is a fundamental technique in biological research and biotechnology. It involves preserving living cells at extremely low temperatures, halting their metabolic activity to maintain their viability and function over extended periods. This method is vital for various scientific and medical applications, allowing for the long-term storage of biological material.
Why Cells Are Frozen
Freezing cells offers several advantages. It allows for the long-term preservation of cell lines, including patient samples and research models, ensuring their availability for future experiments. Cryopreservation helps maintain the genetic stability of cells over time, preventing unwanted changes that can occur with continuous growth and passaging in culture. This technique also reduces the risk of contamination from repeated handling and minimizes phenotypic drift, where cell characteristics change over successive generations. Furthermore, it proves cost-effective by reducing the need for continuous cell culture maintenance.
Getting Ready to Freeze Cells
Careful preparation is important before freezing cells. Cells should be healthy and in an optimal growth phase, such as the log phase, with high viability, typically above 90%. A cryoprotective medium is necessary, usually consisting of cell culture medium supplemented with a cryoprotectant. Dimethyl sulfoxide (DMSO) or glycerol are common choices, typically used at a concentration of 10%.
Sterile cryovials are used to store the cell suspension, and aseptic technique must be maintained throughout the process to prevent contamination. A controlled-rate freezing device, such as a programmable freezer or an isopropanol-containing container like a “Mr. Frosty,” ensures a gradual temperature decrease. This controlled cooling is important for minimizing damage to the cells.
The Cell Freezing Process
The cell freezing process involves several steps. Initially, cells are harvested, such as by enzymatic digestion, and then concentrated by centrifugation. After harvesting, the cells are counted, and their viability is assessed, typically using trypan blue exclusion, to ensure a healthy starting population. The cell pellet is then gently resuspended in the prepared cryoprotective medium at a specific concentration, often between 2 million to 5 million cells per milliliter for many cell types.
The cell suspension is then aliquoted into labeled cryovials, ensuring the vials are tightly sealed to prevent liquid nitrogen from entering. Controlled cooling follows, where the temperature is reduced at approximately 1°C per minute. This slow cooling minimizes the formation of damaging ice crystals within the cells. Once the vials reach approximately -80°C, they are transferred for long-term storage in liquid nitrogen, which maintains temperatures between -135°C and -196°C.
Reviving Frozen Cells
Thawing and recovering frozen cells requires a rapid and gentle approach to preserve their viability. The cryovial is removed from liquid nitrogen storage and immediately placed into a 37°C water bath. Rapid thawing is important to prevent the formation of large ice crystals that can damage cells. The vial should be swirled gently until only a small ice crystal remains.
Once thawed, the cell suspension is gently transferred to a larger volume of fresh, pre-warmed culture medium. This dilution helps to quickly reduce the concentration of the cryoprotectant, which can be toxic to cells at warmer temperatures. The cells are then centrifuged to remove the cryoprotectant-containing medium, and the cell pellet is resuspended in fresh culture medium. Finally, the cells are plated into culture flasks and incubated under appropriate conditions to allow them to recover and resume growth.
Maximizing Cell Viability
Several factors contribute to maximizing cell viability and recovery after cryopreservation. These include starting with healthy cells in their optimal growth phase and high initial viability. Proper cryoprotectant selection and concentration (e.g., 10% DMSO or glycerol) prevents intracellular ice formation and osmotic stress. Adhering to controlled freezing rates (around 1°C per minute) minimizes cellular damage.
Rapid thawing at 37°C avoids recrystallization. Preventing osmotic shock during post-thaw dilution helps maintain cell integrity. Long-term storage in liquid nitrogen is crucial, as prolonged storage at -80°C can lead to viability decline. Post-thaw viability checks assess the process and ensure recovered cell quality.