Cryopreservation is the necessary method of preserving living cells and tissues at extremely low temperatures for long-term storage in research and clinical applications. This technique effectively suspends cellular metabolism, which is essential for maintaining a stable supply of high-quality samples for future use. The primary objective of cryopreservation is not simply to freeze the cells, but to maintain the highest possible post-thaw viability and functionality. Achieving this goal depends entirely on the precision and careful execution of each step in the methodology, from preparation to final recovery.
Cell Health and Preparation Before Freezing
Maximizing cell survival begins long before the actual freezing process with careful attention to the health of the cells being preserved. The cells must be in their logarithmic growth phase, which is the period of rapid, healthy division before the culture becomes too dense. For adherent cells, this typically means harvesting them when they are not fully confluent, often between 70% and 90%.
Using cells that are actively growing ensures they possess the metabolic strength needed to withstand the stresses of freezing and thawing. Before adding the protective freezing medium, the cells must be washed to remove any debris or spent culture media. This washing step is important because residual enzymes or waste products can contribute to cell damage during the ultra-cold storage period.
The final cell concentration is a factor that impacts post-thaw recovery. Most protocols recommend resuspending the cells in the freezing medium at a density between 1 million and 10 million cells per milliliter. A concentration that is too low can result in poor viability, while a concentration that is too high can lead to clumping and uneven exposure to the protective agents.
The Role of Cryoprotective Agents
The process of freezing water poses two main threats to cells: the mechanical damage from ice crystals and the osmotic shock from increased solute concentration. Cryoprotective agents (CPAs) are compounds specifically added to the freezing medium to mitigate these damaging effects. These agents work by lowering the freezing point of the solution and promoting the dehydration of the cell before ice forms.
The most widely used CPA is Dimethyl Sulfoxide (DMSO), typically included at a concentration of 10% in the freezing medium. DMSO is a small molecule that can easily penetrate the cell membrane, which is necessary for it to exert its protective effect inside the cell. Once inside, DMSO interacts with water molecules through hydrogen bonding, which hinders the formation of large, destructive intracellular ice crystals.
While DMSO is highly effective, it is also toxic to cells at warmer temperatures and high concentrations. Alternative permeating CPAs, such as glycerol, are sometimes used, particularly for certain cell types like blood cells. The freezing medium is often prepared with a high concentration of serum, like fetal bovine serum, to provide additional protein-based protection, although defined serum-free media are also available for sensitive or clinical applications.
Managing the Freezing and Storage Process
The controlled reduction of temperature is the procedural step that minimizes the stress of ice formation on the cells. This controlled cooling is designed to allow water to slowly exit the cell, minimizing the chance of ice crystals forming within the cell itself. For most mammalian cells, the optimal cooling rate is approximately -1°C per minute.
This precise rate of cooling is achieved either through a programmable controlled-rate freezer or, more commonly in many laboratories, with a passive freezing device. These passive containers, such as the Mr. Frosty or CoolCell devices, are placed in a -80°C freezer and contain insulation designed to ensure the temperature drops at the target rate of -1°C per minute. The vials are typically left in the -80°C freezer overnight to complete the initial freezing stage.
Once the initial freezing is complete, the vials must be transferred quickly to long-term storage below -130°C to halt all metabolic activity. The most effective long-term storage is in the vapor phase of liquid nitrogen, which maintains a temperature near -196°C. Storage at -80°C is considered short-term, as cell viability will gradually decline over time due to slow biochemical changes and the risk of ice recrystallization.
Maximizing Viability During Thawing and Recovery
The thawing process is a point where cell viability is highly vulnerable, primarily due to the toxicity of the cryoprotective agent and the risk of osmotic shock. Therefore, the thawing must be executed as rapidly as possible to minimize these negative effects. Vials should be transferred immediately from cryogenic storage and placed into a 37°C water bath.
The vial should be gently swirled in the water bath until the contents are just thawed, meaning only a small sliver of ice remains. This rapid warming, often taking only one to two minutes, is necessary to quickly pass the temperature range where ice crystals can reorganize and cause mechanical damage. Prolonged exposure to the 37°C temperature once thawed allows the toxic CPA to begin damaging the cell membranes.
After thawing, the next immediate step is to dilute and remove the CPA, such as DMSO, by transferring the cell suspension to a sterile tube with pre-warmed culture media. The cells are then gently centrifuged to form a pellet, allowing the supernatant containing the toxic agent to be carefully removed. The final step involves resuspending the cells in fresh, pre-warmed media and transferring them to a culture vessel for recovery. The overall success of the process is often confirmed by performing a post-thaw viability assessment, such as using the Trypan Blue exclusion method, to count the proportion of living, intact cells.