Cryoprotectant

A cryoprotectant is a substance used to protect biological tissue from freezing damage (i.e. that due to ice formation). Arctic and Antarctic insects, fish and amphibians create cryoprotectants (antifreeze compounds and antifreeze proteins) in their bodies to minimize freezing damage during cold winter periods. Cryoprotectants are also used to preserve living materials in the study of biology and to preserve food products.

For years, glycerol has been used in cryobiology as a cryoprotectant for blood cells and bull sperm, allowing storage in liquid nitrogen at temperatures around −196 °C. However, glycerol cannot be used to protect whole organs from damage. Instead, many biotechnology companies are researching the development of other cryoprotectants more suitable for such uses. A successful discovery may eventually make possible the bulk cryogenic storage (or "banking") of transplantable human and xenobiotic organs. A substantial step in that direction has already occurred. Twenty-First Century Medicine has vitrified a rabbit kidney to -135 °C with their proprietary vitrification cocktail. Upon rewarming, the kidney was successfully transplanted into a rabbit, with complete functionality and viability, able to sustain the rabbit indefinitely as the sole functioning kidney.^[1]

Mechanism

Cryoprotectants operate by increasing the solute concentration in cells. However, in order to be biologically viable they must easily penetrate and must not be toxic to cells.

Glass transition temperature

Some cryoprotectants function by lowering the glass transition temperature of a solution or of a material. In this way, the cryoprotectant prevents actual freezing, and the solution maintains some flexibility in a glassy phase. Many cryoprotectants also function by forming hydrogen bonds with biological molecules as water molecules are displaced. Hydrogen bonding in aqueous solutions is important for proper protein and DNA function. Thus, as the cryoprotectant replaces the water molecules, the biological material retains its native physiological structure and function, although they are no longer immersed in an aqueous environment. This preservation strategy is most often utilized in anhydrobiosis.

Toxicity

Mixtures of cryoprotectants have less toxicity and are more effective than single-agent cryoprotectants.^[2] A mixture of formamide with DMSO (dimethyl sulfoxide), propylene glycol, and a colloid was for many years the most effective of all artificially created cryoprotectants. Cryoprotectant mixtures have been used for vitrification (i.e. solidification without crystal ice formation). Vitrification has important applications in preserving embryos, biological tissues and organs for transplant. Vitrification is also used in cryonics, in an effort to eliminate freezing damage.

Conventional

Conventional cryoprotectants are glycols (alcohols containing at least two hydroxyl groups), such as ethylene glycol ^{[citation needed]}, propylene glycol and glycerol. Ethylene glycol is commonly used as automobile antifreeze; while propylene glycol has been used to reduce ice formation in ice cream. Dimethyl sulfoxide (DMSO) is also regarded as a conventional cryoprotectant. Glycerol and DMSO have been used for decades by cryobiologists to reduce ice formation in sperm,^[3] oocytes,^[4] and embryos that are cold-preserved in liquid nitrogen. Cryoconservation of animal genetic resources is a practice that involves conventional cryoprotectants to store genetic material with the intention of future revival. Trehalose is non-reducing sugar produced by yeasts and insects in copious amounts. Its use as a cryoprotectant in commercial systems has been patented widely.

Examples in nature

Arctic fish use antifreeze proteins, sometimes appended with sugars, as cryoprotectants.

Insects

Further information: Antifreeze protein and Threitol

Insects most often use sugars or polyols as cryoprotectants. One species that uses cryoprotectant is Polistes exclamans (a wasp). In this species, the different levels of cryoprotectant can be used to distinguish between morphologies.^[5]

Amphibians

Cold-adapted arctic frogs, such as wood frogs, and some other ectotherms in polar and subpolar regions naturally produce glucose,^[6] but southern brown tree frogs and Arctic salamanders create glycerol in their livers to reduce ice formation.

When glucose is used as a cryoprotectant by arctic frogs, massive amounts of glucose are released at low temperature and a special form of insulin allows for this extra glucose to enter the cells. When the frog rewarms during spring, the extra glucose must be rapidly eliminated, but stored.

Food preservation

Cryoprotectants are also used to preserve foods. These compounds are typically sugars that are inexpensive and do not pose any toxicity concerns. For example, many (raw) frozen chicken products contain a sucrose and sodium phosphates solution in water.

Common

DMSO Ethylene glycol Glycerol

2-Methyl-2,4-pentanediol (MPD) Propylene glycol Sucrose Trehalose Heavy water [7]

See also

• Antifreeze protein
• Cryoconservation of animal genetic resources
• Cryoconservation of plant genetic resources
• Cryopreservation
• Cryostasis (clathrate hydrates)
• List of emerging technologies
• Lyophilization

References

1. Fahy GM; Wowk B; Pagotan R; Chang A; et al. (2009). "Physical and biological aspects of renal vitrification". Organogenesis. 5 (3): 167–175. doi:10.4161/org.5.3.9974. PMC 2781097. PMID 20046680.
2. Best, BP (2015). "Cryoprotectant Toxicity: Facts, Issues, and Questions". Rejuvenation Research. 18 (5): 422–436. doi:10.1089/rej.2014.1656. PMC 4620521. PMID 25826677.
3. Imrat, P.; Suthanmapinanth, P.; Saikhun, K.; Mahasawangkul, S.; Sostaric, E.; Sombutputorn, P.; Jansittiwate, S.; Thongtip, N.; et al. (February 2013). "Effect of pre-freeze semen quality, extender and cryoprotectant on the post-thaw quality of Asian elephant (Elephas maximus indicus) semen" (PDF). Cryobiology. 66 (1): 52–59. doi:10.1016/j.cryobiol.2012.11.003. hdl:2263/42468. PMID 23168056.
4. Karlsson, Jens O.M.; Szurek, Edyta A.; Higgins, Adam Z.; Lee, Sang R.; Eroglu, Ali (February 2014). "Optimization of cryoprotectant loading into murine and human oocytes". Cryobiology. 68 (1): 18–28. doi:10.1016/j.cryobiol.2013.11.002. PMC 4036103. PMID 24246951.
5. J.E. Strassmann; R.E. Lee Jr.; R.R. Rojas & J.G Baust (1984). "Caste and sex differencesin cold-hardiness in the social wasps, Polistes annularis and P. exclamans". Insectes Sociaux. 31 (3): 291–301. doi:10.1007/BF02223613. S2CID 39394207.
6. Larson, D. J.; Middle, L.; Vu, H.; Zhang, W.; Serianni, A. S.; Duman, J.; Barnes, B. M. (15 April 2014). "Wood frog adaptations to overwintering in Alaska: New limits to freezing tolerance". Journal of Experimental Biology. 217 (12): 2193–2200. doi:10.1242/jeb.101931. PMID 24737762.

7. Urmatskikh A.V. "Method of cryopreservation of cells, organs, tissues and organisms." RU 2804972 C2, 04.05.2022.