Cryopreservation is the process of preserving cells, tissues, and other biological materials by cooling them to very low temperatures. During this process, it’s essential to avoid the formation of ice crystals, which otherwise destroy cellular membranes and make them unviable. In order to reduce the risk of ice crystal formation, cryoprotectant agents are used.
How do cryoprotective agents work?
Cryoprotectants, also known as cryoprotective agents or CPAs, are a type of medical-grade antifreeze introduced through a process called perfusion. They protect against ice formation and lead to vitrification, which is the transformation of a substance into a glass-like amorphous state. Currently, cryopreservation is successfully used in the storage of stem cells, assisted reproduction technology, and even for the preservation of certain animals, plants, and seeds [4]. Some of the most prevalent CPAs used today include dimethylsulfoxide (DMSO) and ethylene glycol.
Unfortunately, both of these agents are toxic to a degree, thus additional damage would need to be repaired before the warming of complex cellular organisms. Their toxicity also requires that they be administered at near-freezing temperatures.
This is one reason cryopreservation cannot yet be used for organ storage and donation nor can reanimation occur. Yet, 60% of donor hearts and lungs are not used or transplanted due in part to exceeding their maximum hypothermic preservation times, and this waste could be overcome with cryopreservation [1]. One of the main barriers to this is insufficient time for CPAs to penetrate the cells prior to toxicity.
Exciting new research recently published in the Journal of Materials Chemistry B demonstrates the potential for increased efficacy of new cryoprotectants in regard to organ transplants. Lead researcher, Dr. Saffron Bryant, and his team at RMIT University found that using deep eutectic solvents as cryoprotectants reduces toxicity and improves the outcome of cryoprotectant solutions on cellular viability post-thaw. This is one of the first and only times this class of solvents has been systematically tested for cryopreservation of mammalian cells [4].
What are deep eutectic solvents?
Deep eutectic solvents (DESs) are mixtures of hydrogen bond donors and acceptors that have melting points much lower than the individual components [2][3]. They combine previously studied cryoprotectants with other organic components to alter cellular structures and reactivity. This allows for extensive fine-tuning of chemical structures for unique applications without the risk of toxicity.
In this study, six DESs were explored for their cryoprotective capacity towards mammalian cells, which included various ratios of mixtures of galactose (Gal) or glycerol (Gly) with either choline chloride (ChCl), betaine (Bet), or proline (Prol) [4]. They were tested for their toxicity, thermal behavior, glass transition, and, ultimately, their cryoprotective capacity against four different human cell types, including skin and brain cells [4].
How deep eutectic solvents affect cryopreservation
Results demonstrated that the combination of proline and glycerol (Prol-Gly) was almost as effective as DMSO without a significant reduction in cellular viability post-thaw, even with an extended incubation prior to freezing [4].
Their results support the current practice of combining cryoprotective agents. These findings further highlight the importance of using multicomponent systems to reduce toxicity and improve the long-term viability of cryopreservation rather than single cryoprotective agents.
This is still a potentially new application and more research is required before deep eutectic solvents could be used for the cryopreservation of tissues and organs. This study does, however, give insight as to the potential for thousands of potentially new cryoprotectant agents, which may eventually result in improved technologies and processes that could revolutionize the way we view Biostasis.
Specifically, these findings could eventually lead to the further development of new cryoprotective agents that could be tailored to target specific cell types [4]. In due course, this could result in effective organ storage and even new advancements in cryopreservation and revival.
Conclusion
This research has only just begun to explore the effect of new CPAs on single cells. When you view the entire organ (or organism) as a whole, it becomes a much more complicated process. This application may not be fit for human cryopreservation, as the new cryoprotectants identified in the study seem to have a low penetration rate (non-penetrating CPA) compared to currently used CPAs. However, the results provide exciting implications regarding the future of research and development in cryonics.
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References
[1] Manuchehrabadi, N., Gao, Z., Zhang, J., Ring, H. L., Shao, Q., Liu, F., McDermott, M., Fok, A., Rabin, Y., Brockbank, K. G. M., Garwood, M., Haynes, C. L., & Bischof, J. C. (2017, March 1). Improved tissue cryopreservation using inductive heating of magnetic nanoparticles. Science translational medicine. Retrieved July 13, 2022, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470364/
[2] Abbott, A. P., Capper, G., Davies, D. L., Rasheed, R. K., & Tambyrajah, V. (2002, November 26). Novel solvent properties of choline chloride/urea mixtures. Chemical Communications. Retrieved July 13, 2022, from https://pubs.rsc.org/en/content/articlelanding/2003/cc/b210714g
[3] Bryant, S. J., Christofferson, A. J., Greaves, T. L., McConville, C. F., Bryant, G., & Elbourne, A. (2021, October 29). Bulk and interfacial nanostructure and properties in deep eutectic solvents: Current perspectives and Future Directions. Journal of Colloid and Interface Science. Retrieved July 13, 2022, from https://www.sciencedirect.com/science/article/abs/pii/S0021979721018415?via%3Dihub
[4] Bryant, S. J., Awad, M. N., Elbourne, A., Christofferson, A. J., Martin, A. V., Meftahi, N., Drummond, C. J., Greaves, T. L., & Bryant, G. (2022, May 31). Deep eutectic solvents as cryoprotective agents for mammalian cells. Journal of Materials Chemistry B. Retrieved July 13, 2022, from https://pubs.rsc.org/en/content/articlelanding/2022/TB/D2TB00573E
