- Cryoprotective agents (CPAs) are substances added to cryopreservation media to reduce cellular and structural damage associated with cooling, freezing, and thawing of biological materials, including cells, tissues, gametes, embryos, and, in some applications, organs.
- CPAs are critical in cryopreservation, a process that involves storing biological samples at ultra‑low temperatures (−150 °C to −196 °C) with the aim of maintaining them in a viable state so they can be revived when required.
- A successful cryopreservation outcome relies not merely on achieving an ultra‑low storage temperature but on carefully controlling the physical and biological events that unfold during both cooling and warming. These transitional phases are critical, as cells are vulnerable to lethal ice formation, damaging osmotic shifts, and structural disruption of cellular membranes and organelles.
- The presence of CPAs helps living materials survive the freeze–thaw cycle. Without CPAs, ice crystals can form and cause mechanical damage as well as osmotic imbalance, ultimately leading to membrane rupture, cell shrinkage, and loss of cellular function.
- CPAs protect biological materials through multiple, overlapping mechanisms rather than a single mode of action. Depending on the CPA and cryopreservation protocol, they can reduce intracellular ice formation, modify water activity and ice nucleation/crystal growth, control cellular dehydration, alter solution viscosity, and stabilize cellular membranes and proteins. CPAs therefore help balance the competing risks of intracellular ice formation, excessive dehydration, osmotic injury.
- CPAs are commonly classified according to their ability to cross cell membranes as permeating (penetrating) and non-permeating (non-penetrating) CPAs. This classification is useful but not absolute, because membrane permeability varies with the CPA, temperature, membrane properties, and biological material being preserved.
- Permeating CPAs, including dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propylene glycol (1,2-propanediol), can enter cells and modify the intracellular aqueous environment. Their intracellular action reduces the probability of lethal intracellular ice formation and helps regulate water movement during cooling. They are widely used in cell, gamete, and embryo cryopreservation, although the optimal CPA, concentration, exposure time, and cooling/warming protocol are highly dependent on the biological material.
- Non-permeating CPAs generally remain predominantly in the extracellular compartment and include sugars such as sucrose and trehalose, as well as some polymers and macromolecules such as polyvinylpyrrolidone (PVP) and hydroxyethyl starch (HES). By increasing extracellular osmolality, they promote controlled water efflux from cells and thereby reduce intracellular water available for ice formation. They may also contribute to membrane and protein stabilization. The specific protective mechanism depends on the CPA and formulation.
- CPA selection is highly material- and protocol-specific. For example, DMSO is widely used for mammalian cells, including hematopoietic and other stem/progenitor cells; glycerol has long been used for sperm and red blood cell cryopreservation; and ethylene glycol, often in combination with other CPAs, is widely used in oocyte and embryo vitrification.
- Although CPAs are essential for many cryopreservation protocols, CPA toxicity is an important limitation. Permeating CPAs such as DMSO can produce concentration- and exposure-time-dependent cytotoxicity and osmotic stress. CPA loading and removal must therefore be carefully controlled, particularly for sensitive cells and vitrification protocols. Post-thaw CPA removal is often performed progressively to minimize osmotic injury and residual toxicity.
- Recent developments aim to reduce CPA toxicity and improve recovery by combining conventional CPAs with serum-free and xeno‑free formulations, optimized CPA mixtures, controlled cooling and warming, and novel ice-modulating materials. These approaches are particularly relevant to cell therapy, regenerative medicine, reproductive medicine, tissue engineering, and biobanking, where reproducible recovery, clinical compatibility, and scalability are important.
Further reading:
- Fuller, B.J., 2004. Cryoprotectants: the essential role of toxicity in cryopreservation. Cryo-Letters. 25(6), 375–388.
- Elliott, G.D., et al., 2017. Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures. Cryobiology. 74-91. DOI: 10.1016/j.cryobiol.2017.04.004, PMID: 28428046, ScienceDirect (Download PDF), Core (Download PDF)
- Best, B. P., 2015. Cryoprotectant Toxicity: Facts, Issues, and Questions. Rejuvenation Res. 18(5), 422-436. DOI: 10.1089/rej.2014.1656, PMID: 25826677, PMCID: PMC4620521 (Download PDF), Sagepub (Download PDF)
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Last updated: 6th August 2026
