4 Tips for Successful Cryopreservation

Cryopreservation is a critical procedure that can help you to conserve biological material for long periods of time. Although it is a very efficient method, it involves reagents and techniques that can fail if not done correctly. In cryopreservation, the aim is to use low temperatures to preserve structurally intact living cells/ tissues. Proper equipment must be in place to ensure consistency, reproducibility, and sterility. The correct choice and amount of cryoprotectant agent must be added at the correct temperature, and a controlled rate of freezing must be applied prior to a standardized method of cryogenic storage. The goal of this guide is to help your cells/tissues to succeed the thermodynamic journey from the 37°C incubator to the -196°C liquid nitrogen storage tank, and live long and prosper.

Select the adequate medium

The first step to ensure the successful preservation of the cells/tissues is to find the adequate medium. In other words, you must check that you are using the right medium for your cells/tissues.

Cryoprotective Agents

Cryoprotectants safeguard slowly frozen cells/tissues by 4 mechanisms: reducing high salt concentrations, decreasing cell shrinkage, reducing the fraction of the solution that is frozen and/or minimizing intracellular ice formation. The combinations of cryoprotectants may result in additive or synergistic enhancement of cell survival. Glycerol, salts and dimethyl sulfoxide (DMSO) are the most commonly used cryoprotectants. The formation of ice can be eliminated if cryoprotectants are used in extremely high concentrations, at least 50% volume/volume.

Intracellular cryoprotectants with low molecular weights, which will permeate cells/tissues, like glycerol and DMSO at concentrations from 0.5 to 3 molar, are effective in minimizing cell/tissue damage in slowly frozen cells/tissues.

Extracellular cryoprotectants have higher molecular weights, greater than sucrose, which can’t penetrate cells/tissues, like polyvinylpyrrolidone and hydroxyethyl starch. These compounds are more effective at protecting biological systems cooled at rapid rates because they induce vitrification (extracellular glass formation). In some cases, fetal bovine serum (FBS) can be added in mammalian cryopreservation solutions, but it is not a cryoprotectant.

Vitrification

“Vitri” means glass in Greek and it refers to the formation of glass-like structures inside the cells/tissues. Indeed, vitrification is the conversion of a liquid into a glass, and this solidification is due to increased viscosity rather than to crystallization. Preventing freezing requires that the water in a tissue remains liquid during cooling and can be achieved if the samples are vitrified but stored at, or just below, the glass transition temperature.

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Ice Blockers

Some proteins are ice blockers, as they have anti-freezing properties; by binding to the ice crystals in such a way that ice crystallization is inhibited. Synthetic ice blockers have been developed and they may be combined with “natural” proteins and conventional cryoprotectants to improve the preservation of cells/tissues.

Media for Cryopreservation

The appropriate cryoprotective solution can substitute the physiological environment/ medium that the cells/tissue were embedded on. The buffer medium should contain special cryoprotectants essential to minimize freeze-induced injury.

In the pre-freeze phase the cells/tissue should be subjected to cooling, with the purpose of slowing down the metabolism, minimizing ischemic and hypoxic changes and reducing the chemical toxicity of the cryoprotectants.

The samples can be packaged in the pre-cooled cryoprotective solution 0°C to 4°C and then transferred to a similarly pre-cooled cryopreservation chamber. Another option is to use a controlled rate freezer. In this phase, you must take into consideration that the conventional tissue culture media used to nurture cells/tissues at physiological temperatures might not be a suitable medium for exposure at low temperatures. Rapid cooling may be harmful due to thermal shock, therefore preserving the ionic and hydraulic balance within tissues during cooling can be better controlled in media designed to physically restrict these temperature-induced imbalances. The optimal solutions must adjust the ionic balance and raise the osmolality.

Nowadays, ready-to-use solutions such as CryoStemTM are the most reliable, easy to use and safest options. Some benefits of using solutions like this include: being protein-free, works with various media, suitable for freezing cells cultured in both feeder and feeder free conditions and proven high recovery efficiency for human cells. CryoStemTM also contains methylcellulose and DMSO in place of serum, eliminating the risks associated with animal sera and serum-derived products.

Some preparations/cells might need extra formulations. Always read the whole description and recommendations of the reagents that you will employ.

Cooling Rate

The cooling rate may have a significant influence on cell survival. Programmed and uniform cooling rates are crucial for effective long-term storage and to maximize viability of cells/tissues.

The cooling rate may be controlled between 4°C and at least -40°C. Maintaining the cooling uniformity in all the samples is also very important and some devices inject and distribute liquid nitrogen into the chamber by an internal fan, minimizing standard deviation to less than 2°C during a run. Optimal slow cooling conditions resulting in retained cell viability are defined by the cooling rate that permits some cell dehydration without the formation of significant amounts of intracellular ice.

For some mammalian cells the optimal cooling rate lies between 0.3°C and 10°C per minute. Each cell type has a freezing “frame” in which the cooling rate provides optimal cell survival.

Verify the equipment and the freezing method

Be sure you have the right equipment and the corresponding freezing method.

The main benefit of the controlled rate freezing equipment is that the controlled rate preservation for release of the latent heat results in improved post-cryopreservation cell viability.

The most commonly used methods are: liquid nitrogen methods, direct temperature feedback, timed pulse, liquid nitrogen submersion or plunge freezing, and the step down method. Liquid nitrogen is one of the best choices as it is chemically inert, non-flammable and relatively low cost.

The Direct Temperature Feedback method works by injecting liquid nitrogen into the chamber and monitoring the sample temperature while comparing the actual temperature to program chamber temperatures, and it automatically adjusts for low pressure liquid nitrogen supply, freezing rates, or defective solenoid valves.

In the Timed Pulse method, the amount of liquid nitrogen injected is determined by valve size, tank pressure, valve core wear resistance and number of solenoid openings, determined by the temperature-freezing rate programmed.

In the Liquid Nitrogen Submersion or Plunge Freezing, the samples are loaded into a heat block, and that block is submerged into nitrogen. Then, the heater tempers the nitrogen to obtain a controlled freezing rate. It is recommended for small numbers of low volume straws and vials.

In the Step Down freezing method the samples are placed in a refrigerator overnight, transferred to a -70°C freezer for a period of time, and then moved to nitrogen vapor.

Freezing Protocols

It is recommended to optimize the freezing protocol for your cells/tissues testing different times and temperatures to determine the steps required to obtain the desired level of cell viability or tissue function. The manufacturers of the selected device or medium might recommend protocols, or programs, that may produce acceptable viability and they are a good starting point.

A generally used protocol for a 2 ml sample size, suggests a 1°C rate from nucleation to -40°C and a 10°C per minute cooling rate to a -90°C end temperature.

Storing Cryopreserved Samples

As a rule of thumb, the lower the temperature, the longer the viable storage period. Some samples may be stored at -70°C for months or years, but the chemical reactions responsible for cellular deterioration are not completely stopped at this temperature. Samples at temperatures below -130°C may be stored for millennia.

Storage Phases

The samples may be stored in the vapor phase or in the liquid phase. Liquid phase storage offers a uniform temperature of -196°C. The cons of this method are that packaging materials used (cryovials) may leak and allow liquid nitrogen to enter the package and, because the nitrogen expands to gas as it is warmed, it may cause an explosion unless the gas is allowed to escape when the samples are recovered. Tip: opening the vial lid immediately to diffuse the pressure alleviates this threat.

Vapor phase storage offers a temperature gradient, and not a uniform temperature. Storage in the vapor phase reduces the possibility of cross contamination, but doesn’t have as much of a safety margin in terms of storage time.

Storage System or Container

The main four factors to select the best container are the cost (price, quality, warranty), consumption (as liquid nitrogen loss rate), sample capacity, and control (temperature, autofill, and monitoring).

Safety and quality control

Since nitrogen gas is colorless, odorless and tasteless, it cannot be detected by the human senses, and might cause dizziness and lead to unconsciousness and death.

Improperly sealed containers may explode. Placing containers in vapor phase nitrogen for several hours before immersing them in liquid nitrogen minimizes the risk of explosion.

The risk of sample contamination and user contamination by hazardous samples can be avoided by opening the container in a safe cabinet.

Safety Recommendations Users should wear a protective face covering, insulated gloves, and long-sleeved clothing to prevent exposure to liquid nitrogen. Always use containers designed for low temperature liquids, don’t seal or prevent liquid nitrogen from venting. Use solid rods as measurement sticks, use liquid nitrogen in well-ventilated areas only and don’t overfill containers. Protocols for decontamination should be developed for immediate corrective action. Always update the information of each rack, box and well with all the sample information: type, date, person responsible, etc.

Recovery protocol

One of the major challenges is how to get the cells/tissues back to life in the best shape. In the case of mammalian embryos slow warming is essential for survival, because it allows sufficient time for cell rehydration and gradual loss of accumulated solutes. For cells cryopreserved that are warmed slowly, the small ice crystal nuclei tend to grow by recrystallization, causing cell damage. The cells can survive rapid thawing because they accumulated relatively little solute during rapid cooling, and recrystallization has little opportunity to occur during rapid warming. A good practice of diluting the cryoprotectant incrementally, at appropriate time intervals, to maintain cell volume below the threshold for damage and diluting the cryoprotectant in the presence of a non-permeating solute (sucrose), prevents cell swelling as solutes diffuse out of the cells.

References

1. Nishiyama, Y., Iwanami, A., Kohyama, J., Itakura, G., Kawabata, S., Sugai, K., Nishimura, S., Kashiwagi, R., Yasutake, K., Isoda, M., Matsumoto, M., Nakamura, M. and Okano, H. “Safe and efficient method for cryopreservation of human induced pluripotent stem cell-derived neural stem and progenitor cells by a programmed freezer with a magnetic field.” Neuroscience Research Volume 107, June 2016, Pages 20-29 https://doi.org/10.1016/j. neures.2015.11.011 
2. K. Imaizumi, N. Nishishita, M. Muramatsu, T. Yamamoto, C. Takenaka, S. Kawamata, K. Kobayashi, S. Nishikawa, T. Akuta “A simple and highly effective method for slow-freezing human pluripotent stem cells using dimethyl sulfoxide, hydroxyethyl starch and ethylene glycol” PLOS ONE, 9 (2014), p. e88696
3. Brockbank, K. G. M. “Essentials of cryobiology.” In Principles of Autologous, Allogeneic, and Cryopreserved Venous Transplantation. (Ed. K. G. M. Brockbank), RG Landes Company, Austin, TX (Medical Intelligence Unit Series) Springer-Verlag, 91-102, 1995. 
4. Brockbank, K. G. M. “Method for cryopreserving blood vessels.” U.S. Patent 5,145,769, and Patent 5,158,867, 1992. 
5. Whittingham, D. G., S. P. Leibo, and P. Mazur. “Survival of mouse embryos frozen to -196C and -269C.” Science, NY 178: 411-14, 1972. 
6. Wolfinbarger L., M. Adam, P. Lange, and J. F. Hu. “Microfractures in cryopreserved heart valves: valve submersion in liquid nitrogen revisited.” Applications of Cryogenic Technology, Plenum Press, 10: 227-233, 1991.

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