Cryopreservation

• Cryopreservation (derived from the Greek kryos, meaning “frost” or “icy cold,” and the Latin preservare, meaning “to keep intact”) is the long-term storage of viable biological materials at ultra-low cryogenic temperatures, typically below −135 °C (138 K), which corresponds to the glass transition temperature (T(_g)) of pure water.
• At or below this threshold, molecular motion becomes negligible, effectively halting enzymatic activity, metabolic processes, and other biochemical reactions that contribute to cellular aging and degradation.
• By suspending biological time, cryopreservation procedures aim to preserve the structural integrity, genomic stability, and physiological viability of biological materials, including cells, tissues, and organns. The objective is to enable long‑term storage and subsequent recovery without loss of viability or functional capacity following thawing.
  
• Freezing is generally detrimental to living cells because it creates conditions that interfere with cell survival, including ice formation, extreme osmotic shifts, and oxidative stress. These factors can damage membranes, proteins, and intracellular structures during both the freezing and thawing process. Read more about Detrimental Effects of Freezing on Cells and Tissues
• Cryopreservation protocols are designed to minimize freezing-induced cellular injury through the combined use of controlled cooling strategies and cryoprotective agents. 
• Cryoprotective agents are chemical additives that modify the thermodynamic and kinetic behaviour of water, thereby reducing cryoinjury. They depress the freezing point, increase solution viscosity, inhibit ice nucleation and crystal growth, and stabilise cellular membranes and proteins during freezing and thawing. Collectively, these effects reduce the extent of ice formation, limit freeze‑concentration‑induced osmotic stress, and promote an amorphous, glass‑like solidification that occurs without crystalline ice.
  
• Cryopreservation has been successfully applied across a broad spectrum of biological materials, ranging from individual cells, such as spermatozoa, oocytes, embryos, stem cells, and blood cells, to increasingly complex multicellular tissues.
• The optimal cryopreservation strategy depends on the biological material’s size, structural organisation, cellular composition, membrane properties, and intended application, as these factors collectively determine the material’s complexity and strongly influence preservation success.
• Isolated cells are generally more amenable to cryopreservation because of their small size, high surface-area-to-volume ratio, relatively uniform cooling environment, and efficient cryoprotectant exchange. In contrast, multicellular tissues and organs present substantial biophysical challenges arising from complex architecture, heterogeneous cell populations, limited cryoprotectant diffusion, and non-uniform heat and mass transport. Preserving entire organs for transplantation remains a significant challenge in the field.
• Classical cryopreservation procedures involve the slow cooling of biological samples in the presence of low concentrations of cryoprotectants to minimize ice crystal formation, thereby reducing cellular damage. This method is commonly used for the preservation of sperm, blood cells, and various types of tissues. For instance, sperm banks utilize slow freezing techniques to maintain the viability of sperm for future use in assisted reproductive technologies. 
• In contrast, recent advancements in cryopreservation have introduced vitrification, which employs high concentrations of cryoprotectants and involves rapid cooling. This process transforms biological samples into a glass-like state without ice crystal formation, making it particularly effective for the preservation of oocytes, embryos, and certain tissues. Vitrification has become increasingly popular in fertility clinics due to its ability to enhance survival rates upon thawing.
  
• Although cryopreservation is a human‑developed technique, certain organisms have evolved natural freeze tolerance that allows them to survive even when their bodies freeze in their natural habitat. Species such as the wood frog (Lithobates sylvaticus/Rana sylvatica), gall fly larvae (Eurosta solidaginis), and hatchling painted turtles (Chrysemys picta) can endure the formation of ice within their tissues through specialised biochemical and physiological adaptations. Studying these natural examples can provide valuable insights for improving cryopreservation techniques. (read more: Natural Cryopreservation)
• Overall, cryopreservation is a cornerstone technology in reproductive medicine, regenerative biology, conservation, biotechnology, and clinical cell therapy. By understanding the physics of ice formation, the chemistry of solute exclusion, and the biology of cellular stress responses, researchers continue to refine preservation techniques that extend the boundaries of what can be stored, revived, and used in future scientific and medical applications. (read more: Applications of Cryopreservation) 

Further reading:

• Baust J.G., Gao, D., Baust, J.M. 2009. Cryopreservation: An emerging paradigm change. Organogenesis. 5(3):90-6. DOI: 10.4161/org.5.3.10021. PMID: 20046670, PMCID: PMC2781087 (Download PDF), Tandfonline (Download PDF)
• Jang, T. H. et al., 2017. Cryopreservation and its clinical applications. Integrative Medicine Research, 6 (1), 12–18. DOI: 10.1016/j.imr.2016.12.001, PMID-28462139, PMCID: PMC5395684 (Download PDF), Sciencedirect (Download PDF)
• Meneghel, J., Kilbride, P., & Morris, G. J., 2020. Cryopreservation as a key element in the successful delivery of cell-based therapies—A review. Frontiers in Medicine, 7, 592242. DOI: 10.3389/fmed.2020.592242, PMID-33324662, PMCID: PMC7727450 (Download PDF), Frontiersin (Download PDF)

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Last updated: 6th August 2026