Cryopreservation is the process of freezing biological entity at ultra-low temperatures (-196oc) while preserving the structural and functional integrity of cells. Cryopreservation helps to prevent maintenance of cell cultures for extended durations beyond requirement by quarantining of the donor cells. It thereby helps in cost-cutting by conserving the culture media and plasticware used. It further protects the epigenetic signature of the cells that are lost due to multiple passages. Moreover, it can help in the utilization of the same batch of cells at a specific passage number for multiple uses especially when the cells are limited or precious. However, similar to other culture techniques, optimization of the entire cryopreservation process is essential to maintaining cell viability and functionality.
Key Considerations For Cryopreservation:
Optimal Freezing Media
Freezing media usually consist of a combination of cryoprotectants, serum and culture media. Cryoprotectants act by dissolving in water to depress its freezing point and thereby inhibiting ice crystal formation. This also reduces the salt concentration which otherwise causes osmotic stress to cells. The most commonly used cryoprotectants are DMSO and glycerol which are used along with Fetal bovine serum (FBS). However, for clinical applications, due to concerns with immunogenicity, serum-free and xeno-free media are recommended with alternatives such as Human albumin. More recent alternatives are platelet lysates and trehalose. Many commercial freezing media are also available off-the-shelf that may be suitable for specific cell types. For instance, sensitive cultures such as pluripotent stem cells tend to differentiate in the presence of serum and require chemically defined commercial compositions for freezing.
Freezing Protocol
Although protocols for cryopreservation have been very widely reported, it is recommended to optimize the freezing protocol depending on the cell type of interest to maximize viability and functionality post-thawing.
Rate Of Freezing For Cryopreservation
A controlled rate of freezing before long-term storage can maximize cell survival. The optimal rate of cooling must be a balance between very rapid and very slow cooling to prevent dehydration or ice crystallization. Slow cooling substitutes the intracellular water content with cryoprotectant and prevents cell damage. On the other hand, fast cooling minimizes the dehydration rates. Readily available Freezing containers usually ensure a cooling rate of -1oc/min.
Thawing
Rate of thawing is as critical as the rate of freezing for optimal recovery of cells. It is preferred to ensure as rapid warming as possible to reduce the exposure time of cells to intra-cellular salts. The most common practice is to thaw cells in a 37oc water bath which may be coupled with continuous stirring for the uniform thermal gradient. Warming rate may be controlled by automated thawing systems. Furthermore, it is essential to dilute out the cryoprotectant from the cells by centrifugation and resuspension in cryoprotectant free media. Presence of DMSO can be toxic to the cells even in very minute quantities, therefore, Rho-kinase (ROCK) inhibitors may be added to the culture media post-thaw to prevent apoptosis. Additives such as sucrose are known to improve revival rates by cell swelling. Here the challenge is to restore the physiological processes of the cells upon thawing.
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