The objective of cryopreservation is to minimize damage to biological materials, including tissues, mammalian cells, bacteria, fungi, plant cells, and viruses, during low temperature freezing and storage. When performed correctly and accounting for cell and tissue specific criteria, cryopreservation can provide a continuous source of tissues and genetically stable living cells for a variety of purposes, including research and biomedical processes. This article will describe the workflow, methods and equipment used for freezing, storage and thawing samples.
A highly specialized science
As the knowledge base has grown and scientists know more and more about the harm the freezing process can cause, the science of preserving and reanimation of cells has become highly specialized based on factors such as the type of cells, vial or bag volume, throughput, and convenience. Each freezing method available has distinct advantages and limits to their use. This article will look to explore and explain those freezing methods so that a logical choice can be made that best suits the above factors as found in your lab.
Workflow: Samples are grown, collected, vitrified, labeled and stored, retrieved, and analyzed
A simple workflow for cryopreservation looks like this:
The most important stages of the workflow to the survival of samples is the vitrification*, storage, and thawing.
*Vitrification is the process of cooling where the water in the tissue becomes glass rather than crystals. Glass is a liquid that is too cold (too viscous) to flow. In other words vitrification is solidification due to increased viscosity rather than crystallization.
Vitrification Methods: Controlled Rate versus Rapid Freezing
The goal of the cryopreservation process is to avoid crystallization and dehydration of the cell. As the freeze point temperature is approached, ice crystals can form outside of the cell creating an osmotic imbalance with the cell. This imbalance causes water to exit the cell through osmosis resulting in cellular dehydration. Excessive dehydration will create nonviable cells. The detrimental effects of dehydration and crystallization can be minimized by doing the following:
• Controlling the cooling rate
• Using cryoprotective agents
• Maintaining appropriate storage temperatures
• Controlling the thawing rate
All of these events interact to influence the viability of thawed cells.
Controlled Rate Freezing
Cooling rate is known to have a significant influence on cell survival. Controlled rate freezing before long-term storage maximizes viability for a wide variety of cells. If the cells are sensitive to thermal shock the rate of change from room temperature to 1C to 2C below the solution’s freezing point will have a major effect on viability. Optimized cooling rates that best maintain cell viability are ones that permit some cell shrinkage (dehydration) without the formation of significant amounts of intracellular ice.
There are both active and passive methods of controlled rate freezing. In the active method, as the cell freezing point is approached the cooling rate can be increased to offset the exothermic energy release cause by the latent heat of fusion. Doing so can drastically reduce the cellular damage caused by this release of energy and subsequent temperature spike. In the passive method a consistent cooling rate is maintained by the heat transfer coefficient of the container alone.
In the passive method controlled rate cooling can be accomplished with a ultra low temperature laboratory freezer with an insulated accessory that holds the vials and is designed to limit the heat transfer between the vials and the freezer so that a desirable cooling rate within the vial is achieved.
Cryoprotectants protect controlled rate frozen cells by one or more of the following mechanisms:
• Reducing cell shrinkage at a given temperature
• Reducing the fraction of the solution frozen at a given temperature
• Minimizing intracellular ice formation
Rapid FreezingRapid freezing is a fast, uncontrolled cooling rate where little or no cell shrinkage occurs from osmotically driven dehydration. However, at rapid cooling rates the random formation of intracellular ice occurs. Furthermore, under suboptimal re-warming conditions this intracellular ice may grow and destroy the cells.
Freezing EquipmentThere are a variety of ways to control the cooling rate of sample cells with no one way being the best for every laboratory or application. Controlled rate cooling can be accomplished with an ultra low temperature laboratory freezer with an insulated accessory that holds the vials and is designed to limit the heat transfer between the vials and the freezer so that a cooling rate of 1 °C/minute is achieved within the vial. Or it can be done using a programmed cooling rate that controls the flow of liquid nitrogen (LN2) around the sample vessels. Another way is to use an ultra low temperature recirculating bath programmed with a temperature ramp rate. These two later methods also have the advantage of an adjustable cooling rate.
For rapid freezing a blast freezer or a direct plunge into LN2 can be used.
All the methods mentioned should be studied for which type of cells they work best with along with cost and convenience features that meet the needs of the lab (table 1).
Freezing Equipment table 1
StorageThe temperature at which frozen cells are stored has a major effect on how long they can be stored. Typically, the lower the storage temperature is the longer the viable storage period can be. The impact of having samples cross the glass transition phase and go from liquid to a glass is huge—for any biological sample that is either whole cell or whole tissue, its paramount that storage occurs below glass transition. For intracellular components (proteins, nucleic acids) storage at -70C is fine.
While those samples may be stored at -70C (-94F) for months or even years, the chemical reactions responsible for cellular deterioration are not completely halted at this temperature. Samples at temperatures below -130C (-202F), where it is said that biological time has stopped, may be stored indefinitely.
ThawingThe warming rate of cryopreserved samples can also impact cell viability. Historically the thawing rate has been considered less critical than the controlled rate of cooling. However more and more evidence now exists to support the thawing rate being just as important as the cooling rate.
In general cells that were cooled at a controlled rate, particularly if a cryoprotective agent was used, do better with slow thawing to allow time for the cell to rehydrate and loss of accumulated cryoprotective agent.
Cells that were frozen rapidly generally require rapid thawing to prevent crystallization that would cause cell damage.
ConclusionDifferent types of cells have different requirements for optimum preservation and recovery. For example, extracellular ice formation can be avoided by vitrification and controlled rate freezing, storage temperatures need to be selected based on the anticipated storage period, and crystallization may be avoided during recovery by rapid thawing. Selecting the best cryopreservation equipment for your lab, based on the cell type, budget, and the required features, takes some research of the modern equipment and methods available today.
Additional Thermo Scientific™ references and resource links:
Cryopreservation Guide by Dr. Kelvin G. M. Brockbank, James C. Covault, and Dr. Michael J. Taylor
A guide for Proper Cryogenic Preservation by Frank P. Simione
Cryopreservation and Reanimation of Living Cells by Scott D. Pratt and Dieter Rädler