Cryoprotectant

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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.

Contents

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

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

See also

Related Research Articles

<span class="mw-page-title-main">Cryonics</span> Freezing of a human corpse

Cryonics is the low-temperature freezing and storage of human remains, with the speculative hope that resurrection may be possible in the future. Cryonics is regarded with skepticism within the mainstream scientific community. It is generally viewed as a pseudoscience, and its practice has been characterized as quackery.

<span class="mw-page-title-main">Ethylene glycol</span> Organic compound ethane-1,2-diol

Ethylene glycol is an organic compound with the formula (CH2OH)2. It is mainly used for two purposes, as a raw material in the manufacture of polyester fibers and for antifreeze formulations. It is an odorless, colorless, flammable, viscous liquid. It has a sweet taste, but is toxic in high concentrations. This molecule has been observed in outer space.

<span class="mw-page-title-main">Glycerol</span> Chemical compound widely used in food and pharmaceuticals

Glycerol, also called glycerine or glycerin, is a simple triol compound. It is a colorless, odorless, viscous liquid that is sweet-tasting and non-toxic. The glycerol backbone is found in lipids known as glycerides. It is also widely used as a sweetener in the food industry and as a humectant in pharmaceutical formulations. Because of its three hydroxyl groups, glycerol is miscible with water and is hygroscopic in nature.

<span class="mw-page-title-main">Freezing</span> Phase transition of liquid to solid

Freezing is a phase transition where a liquid turns into a solid when its temperature is lowered below its freezing point. In accordance with the internationally established definition, freezing means the solidification phase change of a liquid or the liquid content of a substance, usually due to cooling.

Cryobiology is the branch of biology that studies the effects of low temperatures on living things within Earth's cryosphere or in science. The word cryobiology is derived from the Greek words κρῧος [kryos], "cold", βίος [bios], "life", and λόγος [logos], "word". In practice, cryobiology is the study of biological material or systems at temperatures below normal. Materials or systems studied may include proteins, cells, tissues, organs, or whole organisms. Temperatures may range from moderately hypothermic conditions to cryogenic temperatures.

<span class="mw-page-title-main">Propylene glycol</span> Chemical compound

Propylene glycol (IUPAC name: propane-1,2-diol) is a viscous, colorless liquid, which is nearly odorless but possesses a faintly sweet taste. Its chemical formula is CH3CH(OH)CH2OH. As it contains two alcohol groups, it is classed as a diol. It is miscible with a broad range of solvents, including water, acetone, and chloroform. In general, glycols are non-irritating and have very low volatility.

<span class="mw-page-title-main">Antifreeze protein</span> Class of peptides which help cells survive freezing conditions

Antifreeze proteins (AFPs) or ice structuring proteins refer to a class of polypeptides produced by certain animals, plants, fungi and bacteria that permit their survival in temperatures below the freezing point of water. AFPs bind to small ice crystals to inhibit the growth and recrystallization of ice that would otherwise be fatal. There is also increasing evidence that AFPs interact with mammalian cell membranes to protect them from cold damage. This work suggests the involvement of AFPs in cold acclimatization.

An antifreeze is an additive which lowers the freezing point of a water-based liquid. An antifreeze mixture is used to achieve freezing-point depression for cold environments. Common antifreezes also increase the boiling point of the liquid, allowing higher coolant temperature. However, all common antifreeze additives also have lower heat capacities than water, and do reduce water's ability to act as a coolant when added to it.

Cold hardening is the physiological and biochemical process by which an organism prepares for cold weather.

<span class="mw-page-title-main">Deicing</span> Process of removing ice, snow, or frost from a surface

Deicing is the process of removing snow, ice or frost from a surface. Anti-icing is the application of chemicals that not only deice but also remain on a surface and continue to delay the reformation of ice for a certain period of time, or prevent adhesion of ice to make mechanical removal easier.

Gregory M. Fahy is a California-based cryobiologist, biogerontologist, and businessman. He is Vice President and Chief Scientific Officer at Twenty-First Century Medicine, Inc, and has co-founded Intervene Immune, a company developing clinical methods to reverse immune system aging. He is the 2022–2023 president of the Society for Cryobiology.

<span class="mw-page-title-main">Insect winter ecology</span> Survival strategies of insects during winter

Insect winter ecology describes the overwinter survival strategies of insects, which are in many respects more similar to those of plants than to many other animals, such as mammals and birds. Unlike those animals, which can generate their own heat internally (endothermic), insects must rely on external sources to provide their heat (ectothermic). Thus, insects persisting in winter weather must tolerate freezing or rely on other mechanisms to avoid freezing. Loss of enzymatic function and eventual freezing due to low temperatures daily threatens the livelihood of these organisms during winter. Not surprisingly, insects have evolved a number of strategies to deal with the rigors of winter temperatures in places where they would otherwise not survive.

<span class="mw-page-title-main">Cryopreservation</span> Process to preserve biological matter

Cryopreservation or cryoconservation is a process where biological material - cells, tissues, or organs - are frozen to preserve the material for an extended period of time. At low temperatures any cell metabolism which might cause damage to the biological material in question is effectively stopped. Cryopreservation is an effective way to transport biological samples over long distances, store samples for prolonged periods of time, and create a bank of samples for users. Molecules, referred to as cryoprotective agents (CPAs), are added to reduce the osmotic shock and physical stresses cells undergo in the freezing process. Some cryoprotective agents used in research are inspired by plants and animals in nature that have unique cold tolerance to survive harsh winters, including: trees, wood frogs, and tardigrades.

Semen extender is a liquid diluent which is added to semen to preserve its fertilizing ability. It acts as a buffer to protect sperm cells from their own toxic byproducts, as well as protecting the sperm from cold shock and osmotic shock during the chilling and shipping process. The extender allows the semen to be shipped to the female, rather than requiring the male and female to be near to each other. Special freezing extender use also allows cryogenic preservation of sperm, which may be transported for use, or used on-site at a later date.

Semen cryopreservation is a procedure to preserve sperm cells. Semen can be used successfully indefinitely after cryopreservation. It can be used for sperm donation where the recipient wants the treatment in a different time or place, or as a means of preserving fertility for men undergoing vasectomy or treatments that may compromise their fertility, such as chemotherapy, radiation therapy or surgery. It is also often used by trans women prior to medically transitioning in ways that affect fertility, such as feminizing hormone therapy and orchiectomies.

<span class="mw-page-title-main">2-Methyl-2,4-pentanediol</span> Chemical compound

2-Methyl-2,4-pentanediol (MPD) is an organic compound with the formula (CH3)2C(OH)CH2CH(OH)CH3. This colourless liquid is a chiral diol. It is produced industrially from diacetone alcohol by hydrogenation. Total European and USA production was 15000 tonnes in 2000.

Freezing tolerance describes the ability of plants to withstand subzero temperatures through the formation of ice crystals in the xylem and intercellular space, or apoplast, of their cells. Freezing tolerance is enhanced as a gradual adaptation to low temperature through a process known as cold acclimation, which initiates the transition to prepare for subzero temperatures through alterations in rate of metabolism, hormone levels and sugars. Freezing tolerance is rapidly enhanced during the first days of the cold acclimation process when temperature drops. Depending on the plant species, maximum freezing tolerance can be reached after only two weeks of exposure to low temperatures. The ability to control intercellular ice formation during freezing is critical to the survival of freeze-tolerant plants. If intracellular ice forms, it could be lethal to the plant when adhesion between cellular membranes and walls occur. The process of freezing tolerance through cold acclimation is a two-stage mechanism:

<span class="mw-page-title-main">Cryoconservation of animal genetic resources</span>

Cryoconservation of animal genetic resources is a strategy wherein samples of animal genetic materials are preserved cryogenically.

Plant cryopreservation is a genetic resource conservation strategy that allows plant material, such as seeds, pollen, shoot tips or dormant buds to be stored indefinitely in liquid nitrogen. After thawing, these genetic resources can be regenerated into plants and used on the field. While this cryopreservation conservation strategy can be used on all plants, it is often only used under certain circumstances: 1) crops with recalcitrant seeds e.g. avocado, coconut 2) seedless crops such as cultivated banana and plantains or 3) crops that are clonally propagated such as cassava, sweet potato.

<span class="mw-page-title-main">Dry shipper</span>

A dry shipper, or cryoshipper, is a container specifically engineered to transport biological specimens at cryogenic temperatures utilizing the vapor phase of liquid nitrogen.

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.

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