Cryo-preservation or cryo-conservation is a process where

organisms In biology, an organism () is any living system that functions as an individual entity. All organisms are composed of cells (cell theory). Organisms are classified by taxonomy into groups such as multicellular animals, plants, and fungi; ...

organelle In cell biology, an organelle is a specialized subunit, usually within a cell, that has a specific function. The name ''organelle'' comes from the idea that these structures are parts of cells, as organs are to the body, hence ''organelle,'' the ...

cells Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Locations * Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery w ...

, tissues,

extracellular matrix In biology, the extracellular matrix (ECM), also called intercellular matrix, is a three-dimensional network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide stru ...

organs In biology, an organ is a collection of tissues joined in a structural unit to serve a common function. In the hierarchy of life, an organ lies between tissue and an organ system. Tissues are formed from same type cells to act together in a fu ...

, or any other biological constructs susceptible to damage caused by unregulated

chemical kinetics Chemical kinetics, also known as reaction kinetics, is the branch of physical chemistry that is concerned with understanding the rates of chemical reactions. It is to be contrasted with chemical thermodynamics, which deals with the direction in wh ...

are preserved by cooling to very low

temperature Temperature is a physical quantity that expresses quantitatively the perceptions of hotness and coldness. Temperature is measured with a thermometer. Thermometers are calibrated in various temperature scales that historically have relied o ...

s (typically using solid

carbon dioxide Carbon dioxide (chemical formula ) is a chemical compound made up of molecules that each have one carbon atom covalently double bonded to two oxygen atoms. It is found in the gas state at room temperature. In the air, carbon dioxide is transpar ...

or using

liquid nitrogen Liquid nitrogen—LN2—is nitrogen in a liquid state at low temperature. Liquid nitrogen has a boiling point of about . It is produced industrially by fractional distillation of liquid air. It is a colorless, low viscosity liquid that is wide ...

). At low enough temperatures, any

enzymatic Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...

or chemical activity which might cause damage to the biological material in question is effectively stopped. Cryopreservation methods seek to reach low temperatures without causing additional damage caused by the formation of ice crystals during freezing. Traditional cryopreservation has relied on coating the material to be frozen with a class of molecules termed

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, antifreeze compounds and antifreeze prot ...

s. New methods are being investigated due to the inherent

toxicity Toxicity is the degree to which a chemical substance or a particular mixture of substances can damage an organism. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a subst ...

of many cryoprotectants. Cryoconservation of animal genetic resources is done with the intention of conservation of the breed.

Natural cryopreservation

Tardigrade Tardigrades (), known colloquially as water bears or moss piglets, are a phylum of eight-legged segmented micro-animals. They were first described by the German zoologist Johann August Ephraim Goeze in 1773, who called them Kleiner Wasserbär ...

s, microscopic multicellular organisms, can survive

freezing 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 o ...

by replacing most of their internal water with a

sugar Sugar is the generic name for sweet-tasting, soluble carbohydrates, many of which are used in food. Simple sugars, also called monosaccharides, include glucose, fructose, and galactose. Compound sugars, also called disaccharides or double ...

called trehalose, preventing it from crystallization that otherwise damages

cell membrane The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment ( ...

s. Mixtures of solutes can achieve similar effects. Some solutes, including salts, have the disadvantage that they may be toxic at intense concentrations. In addition to the water-bear, wood frogs can tolerate the freezing of their blood and other tissues. Urea is accumulated in tissues in preparation for overwintering, and liver glycogen is converted in large quantities to glucose in response to internal ice formation. Both urea and glucose act as "

s" to limit the amount of ice that forms and to reduce

osmotic Osmosis (, ) is the spontaneous net movement or diffusion of solvent molecules through a selectively-permeable membrane from a region of high water potential (region of lower solute concentration) to a region of low water potential (region of ...

shrinkage of cells. Frogs can survive many freeze/thaw events during winter if no more than about 65% of the total body water freezes. Research exploring the phenomenon of "freezing frogs" has been performed primarily by the Canadian researcher, Dr.

Kenneth B. Storey Kenneth B. Storey (born October 23, 1949) is a Canadian scientist whose work draws from a variety of fields including biochemistry and molecular biology. He is a Professor of Biology, Biochemistry and Chemistry at Carleton University in Ottawa, ...

. Freeze tolerance, in which organisms survive the winter by freezing solid and ceasing life functions, is known in a few vertebrates: five species of frogs (''

Rana sylvatica ''Lithobates sylvaticus'' or ''Rana sylvatica'', commonly known as the wood frog, is a frog species that has a broad distribution over North America, extending from the boreal forest of the north to the southern Appalachians, with several nota ...

'', '' Pseudacris triseriata'', ''

Hyla crucifer The spring peeper (''Pseudacris crucifer'') is a small chorus frog widespread throughout the eastern United States and Canada. They prefer permanent ponds due to their advantage in avoiding predation; however, they are very adaptable with respect ...

'', '' Hyla versicolor'', ''

Hyla chrysoscelis Cope's gray treefrog (''Dryophytes chrysoscelis'') is a species of treefrog found in the United States. It is almost indistinguishable from the gray treefrog ('' Dryophytes versicolor''), and shares much of its geographic range. Both species are ...

''), one of salamanders (''

Salamandrella keyserlingii ''Salamandrella keyserlingii'', the Siberian salamander, is a species of salamander found in Northeast Asia. It lives in wet woods and riparian groves. Distribution It is found primarily in Siberia east of the Sosva River and the Urals, in ...

''), one of snakes (''

Thamnophis sirtalis The common garter snake (''Thamnophis sirtalis'') is a species of thamnophis snake, in the natricine subfamily, which is indigenous to North America and found widely across the continent. Most common garter snakes have a pattern of yellow stri ...

'') and three of turtles (''

Chrysemys picta The painted turtle (''Chrysemys picta'') is the most widespread native turtle of North America. It lives in slow-moving fresh waters, from southern Canada to northern Mexico, and from the Atlantic to the Pacific. They have been shown to prefer l ...

'', ''

Terrapene carolina The common box turtle (''Terrapene carolina'') is a species of box turtle with six existing subspecies. It is found throughout the Eastern United States and Mexico. The box turtle has a distinctive hinged lowered shell (the box) that allows it ...

'', ''

Terrapene ornata ''Terrapene ornata'' is a species of North American box turtle sometimes referred to as the western box turtle or the ornate box turtle. It is one of two recognized species of box turtle in the United States, having two subspecies. The second rec ...

''). Snapping turtles ''

Chelydra serpentina The common snapping turtle (''Chelydra serpentina'') is a species of large freshwater turtle in the family Chelydridae. Its natural range extends from southeastern Canada, southwest to the edge of the Rocky Mountains, as far east as Nova Scotia a ...

'' and wall lizards ''

Podarcis muralis ''Podarcis muralis'' (common wall lizard) is a species of lizard with a large distribution in Europe and well-established introduced populations in North America, where it is also called the European wall lizard. It can grow to about in tota ...

'' also survive nominal freezing but it has not been established to be adaptive for overwintering. In the case of ''Rana sylvatica'' one cryopreservant is ordinary glucose, which increases in concentration by approximately 19 mmol/L when the frogs are cooled slowly.

History

One early theoretician of cryopreservation was

James Lovelock James Ephraim Lovelock (26 July 1919 – 26 July 2022) was an English independent scientist, environmentalist and futurist. He is best known for proposing the Gaia hypothesis, which postulates that the Earth functions as a self-regulating sys ...

. In 1953, he suggested that damage to

red blood cell Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "holl ...

s during freezing was due to

stress, and that increasing the salt concentration in a dehydrating cell might damage it. In the mid-1950s, he experimented with the cryopreservation of rodents, determining that hamsters could be frozen with 60% of the water in the brain crystallized into ice with no adverse effects; other organs were shown to be susceptible to damage. Cryopreservation was applied to human materials beginning in 1954 with three pregnancies resulting from the insemination of previously frozen sperm. Fowl sperm was cryopreserved in 1957 by a team of scientists in the UK directed by Christopher Polge. During 1963, Peter Mazur, at

Oak Ridge National Laboratory Oak Ridge National Laboratory (ORNL) is a U.S. multiprogram science and technology national laboratory sponsored by the U.S. Department of Energy (DOE) and administered, managed, and operated by UT–Battelle as a federally funded research and ...

in the U.S., demonstrated that lethal intracellular freezing could be avoided if cooling was slow enough to permit sufficient water to leave the cell during progressive freezing of the extracellular fluid. That rate differs between cells of differing size and water permeability: a typical cooling rate around 1 °C/minute is appropriate for many mammalian cells after treatment with cryoprotectants such as glycerol or dimethyl sulphoxide, but the rate is not a universal optimum. On April 22, 1966, the first human body was frozen—though it had been embalmed for two months—by being placed in liquid nitrogen and stored at just above freezing. The elderly woman from Los Angeles, whose name is unknown, was soon thawed out and buried by relatives. The first human body to be frozen with the hope of future revival was

James Bedford James Hiram Bedford (April 20, 1893 – January 12, 1967) was an American psychology professor at the University of California who wrote several books on occupational counseling. He is the first person whose body was cryopreserved after legal de ...

's, a few hours after his cancer-caused death in 1967. Bedford is the only

cryonics Cryonics (from el, κρύος ''kryos'' meaning 'cold') is the low-temperature freezing (usually at ) and storage of human remains, with the speculative hope that resurrection may be possible in the future. Cryonics is regarded with skepticis ...

patient frozen before 1974 still preserved today.

Temperature

Storage at very low temperatures is presumed to provide an indefinite longevity to cells, although the actual effective life is rather difficult to prove. Researchers experimenting with dried seeds found that there was noticeable variability of deterioration when samples were kept at different temperatures – even ultra-cold temperatures. Temperatures less than the

glass transition point The glass–liquid transition, or glass transition, is the gradual and reversible transition in amorphous materials (or in amorphous regions within semicrystalline materials) from a hard and relatively brittle "glassy" state into a viscous or rubb ...

(Tg) of polyol's water solutions, around , seem to be accepted as the range where

biological activity In pharmacology, biological activity or pharmacological activity describes the beneficial or adverse effects of a drug on living matter. When a drug is a complex chemical mixture, this activity is exerted by the substance's active ingredient or ...

very substantially slows, and , the boiling point of liquid nitrogen, is the preferred temperature for storing important specimens. While

refrigerator A refrigerator, colloquially fridge, is a commercial and home appliance consisting of a thermally insulated compartment and a heat pump (mechanical, electronic or chemical) that transfers heat from its inside to its external environment so th ...

s, freezers, and extra-cold freezers are used for many items, generally the ultra-cold of liquid nitrogen is required for successful preservation of the more complex biological structures to virtually stop all biological activity.

Risks

Phenomena A phenomenon ( : phenomena) is an observable event. The term came into its modern philosophical usage through Immanuel Kant, who contrasted it with the noumenon, which ''cannot'' be directly observed. Kant was heavily influenced by Gottfried W ...

which can cause damage to cells during cryopreservation mainly occur during the freezing stage, and include solution effects,

extracellular This glossary of biology terms is a list of definitions of fundamental terms and concepts used in biology, the study of life and of living organisms. It is intended as introductory material for novices; for more specific and technical definitions ...

ice formation, dehydration, and intracellular ice formation. Many of these effects can be reduced by cryoprotectants. Once the preserved material has become frozen, it is relatively safe from further damage. ; Solution effects: As ice crystals grow in freezing water,

solutes In chemistry, a solution is a special type of homogeneous mixture composed of two or more substances. In such a mixture, a solute is a substance dissolved in another substance, known as a solvent. If the attractive forces between the solvent ...

are excluded, causing them to become concentrated in the remaining liquid water. High concentrations of some solutes can be very damaging. ; Extracellular ice formation: When tissues are cooled slowly, water migrates out of

and

ice Ice is water frozen into a solid state, typically forming at or below temperatures of 0 degrees Celsius or Depending on the presence of impurities such as particles of soil or bubbles of air, it can appear transparent or a more or less opaq ...

forms in the extracellular space. Too much extracellular ice can cause mechanical damage to the cell membrane due to crushing. ; Dehydration: Migration of water, causing extracellular ice formation, can also cause cellular dehydration. The associated stresses on the cell can cause damage directly. ; Intracellular ice formation: While some

organism In biology, an organism () is any living system that functions as an individual entity. All organisms are composed of cells (cell theory). Organisms are classified by taxonomy into groups such as multicellular animals, plants, and ...

s and tissues can tolerate some extracellular ice, any appreciable intracellular ice is almost always fatal to cells.

Main methods to prevent risks

The main techniques to prevent cryopreservation damages are a well-established combination of ''controlled rate and slow freezing'' and a newer flash-freezing process known as ''vitrification''.

Slow programmable freezing

''Controlled-rate and slow freezing'', also known as ''slow programmable freezing (SPF)'', is a technique where cells are cooled to around -196 °C over the course of several hours. Slow programmable freezing was developed during the early 1970s, and eventually resulted in the first human frozen

embryo An embryo is an initial stage of development of a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male spe ...

birth in 1984. Since then, machines that freeze biological samples using programmable sequences, or controlled rates, have been used for human, animal, and cell biology – "freezing down" a sample to better preserve it for eventual thawing, before it is frozen, or cryopreserved, in liquid nitrogen. Such machines are used for freezing oocytes, skin, blood products, embryos, sperm, stem cells, and general tissue preservation in hospitals, veterinary practices and research laboratories around the world. As an example, the number of live births from frozen embryos 'slow frozen' is estimated at some 300,000 to 400,000 or 20% of the estimated 3 million

in vitro fertilization In vitro fertilisation (IVF) is a process of fertilisation where an egg is combined with sperm in vitro ("in glass"). The process involves monitoring and stimulating an individual's ovulatory process, removing an ovum or ova (egg or eggs) ...

(IVF) births. Lethal intracellular freezing can be avoided if cooling is slow enough to permit sufficient water to leave the cell during progressive freezing of the extracellular fluid. To minimize the growth of extracellular ice crystals and recrystallization,

biomaterial A biomaterial is a substance that has been engineered to interact with biological systems for a medical purpose, either a therapeutic (treat, augment, repair, or replace a tissue function of the body) or a diagnostic one. As a science, biomateria ...

s such as alginates,

polyvinyl alcohol Poly(vinyl alcohol) (PVOH, PVA, or PVAl) is a water-soluble synthetic polymer. It has the idealized formula H2CH(OH)sub>''n''. It is used in papermaking, textile warp sizing, as a thickener and emulsion stabilizer in polyvinyl acetate (PVAc) a ...

or chitosan can be used to impede ice crystal growth along with traditional small molecule cryoprotectants. That rate differs between cells of differing size and water permeability: a typical cooling rate of about 1 °C/minute is appropriate for many mammalian cells after treatment with cryoprotectants such as

glycerol Glycerol (), also called glycerine in British English and glycerin in American English, 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 ...

dimethyl sulfoxide Dimethyl sulfoxide (DMSO) is an organosulfur compound with the formula ( CH3)2. This colorless liquid is the sulfoxide most widely used commercially. It is an important polar aprotic solvent that dissolves both polar and nonpolar compounds a ...

(DMSO), but the rate is not a universal optimum. The 1 °C / minute rate can be achieved by using devices such as a rate-controlled freezer or a benchtop portable freezing container. Several independent studies have provided evidence that frozen embryos stored using slow-freezing techniques may in some ways be 'better' than fresh in IVF. The studies indicate that using frozen embryos and eggs rather than fresh embryos and eggs reduced the risk of stillbirth and premature delivery though the exact reasons are still being explored.

Vitrification Vitrification (from Latin ''vitreum'', "glass" via French ''vitrifier'') is the full or partial transformation of a substance into a glass, that is to say, a non-crystalline amorphous solid. Glasses differ from liquids structurally and glasses po ...

Vitrification is a flash-freezing (ultra-rapid cooling) process that helps to prevent the formation of ice crystals and helps prevent cryopreservation damage. Researchers Greg Fahy and William F. Rall helped to introduce vitrification to reproductive cryopreservation in the mid-1980s. As of 2000, researchers claim vitrification provides the benefits of cryopreservation without damage due to ice crystal formation. The situation became more complex with the development of tissue engineering as both cells and biomaterials need to remain ice-free to preserve high cell viability and functions, integrity of constructs and structure of biomaterials. Vitrification of tissue engineered constructs was first reported by Lilia Kuleshova, who also was the first scientist to achieve vitrification of

oocyte An oocyte (, ), oöcyte, or ovocyte is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in a female fetus in the ovary during female gametogenesis. The female ...

s, which resulted in live birth in 1999. For clinical cryopreservation, vitrification usually requires the addition of cryoprotectants before cooling. Cryoprotectants are macromolecules added to the freezing medium to protect cells from the detrimental effects of intracellular ice crystal formation or from the solution effects, during the process of freezing and thawing. They permit a higher degree of cell survival during freezing, to lower the freezing point, to protect cell membrane from freeze-related injury. Cryoprotectants have high solubility, low toxicity at high concentrations, low molecular weight and the ability to interact with water via hydrogen bonding. Instead of

crystal A crystal or crystalline solid is a solid material whose constituents (such as atoms, molecules, or ions) are arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all directions. In addition, macros ...

lizing, the syrupy solution becomes an

amorphous ice Amorphous ice (non-crystalline or "vitreous" ice) is an amorphous solid form of water. Common ice is a crystalline material wherein the molecules are regularly arranged in a hexagonal lattice, whereas amorphous ice has a lack of long-range order ...

—it ''vitrifies''. Rather than a phase change from liquid to solid by crystallization, the amorphous state is like a "solid liquid", and the transformation is over a small temperature range described as the "

glass transition The glass–liquid transition, or glass transition, is the gradual and reversible transition in amorphous materials (or in amorphous regions within semicrystalline materials) from a hard and relatively brittle "glassy" state into a viscous or rubb ...

" temperature. Vitrification of water is promoted by rapid cooling, and can be achieved without cryoprotectants by an extremely rapid decrease of temperature (megakelvins per second). The rate that is required to attain glassy state in pure water was considered to be impossible until 2005. Two conditions usually required to allow vitrification are an increase of viscosity and a decrease in the freezing temperature. Many solutes do both, but larger molecules generally have a larger effect, particularly on viscosity. Rapid cooling also promotes vitrification. For established methods of cryopreservation, the solute must penetrate the cell membrane in order to achieve increased viscosity and decrease the freezing temperature inside the cell. Sugars do not readily permeate through the membrane. Those solutes that do, such as DMSO, a common cryoprotectant, are often toxic in intense concentration. One of the difficult compromises of vitrifying cryopreservation concerns limiting the damage produced by the cryoprotectant itself due to cryoprotectant toxicity. Mixtures of cryoprotectants and the use of ice blockers have enabled the

21st Century Medicine 21st Century Medicine (21CM) is a California cryobiological research company which has as its primary focus the development of perfusates and protocols for viable long-term cryopreservation of human organs, tissues and cells at temperatures belo ...

company to vitrify a

rabbit Rabbits, also known as bunnies or bunny rabbits, are small mammals in the family Leporidae (which also contains the hares) of the order Lagomorpha (which also contains the pikas). ''Oryctolagus cuniculus'' includes the European rabbit speci ...

kidney The kidneys are two reddish-brown bean-shaped organs found in vertebrates. They are located on the left and right in the retroperitoneal space, and in adult humans are about in length. They receive blood from the paired renal arteries; blood ...

to −135 °C with their proprietary vitrification mixture. Upon rewarming, the kidney was transplanted successfully into a rabbit, with complete functionality and viability, able to sustain the rabbit indefinitely as the sole functioning kidney. In 2000,

FM-2030 FM-2030 (originally born as Fereidoun M. Esfandiary; fa, فریدون اسفندیاری; October 15, 1930 – July 8, 2000) was a Belgian-born Iranian-American author, teacher, transhumanist philosopher, futurist, consultant, and Olympic athlet ...

became the first person to be successfully vitrified posthumously.

Persufflation

Blood can be replaced with inert noble gases and/or metabolically vital gases like

oxygen Oxygen is the chemical element with the symbol O and atomic number 8. It is a member of the chalcogen group in the periodic table, a highly reactive nonmetal, and an oxidizing agent that readily forms oxides with most elements as wel ...

, so that organs can cool more quickly and less antifreeze is needed. Since regions of tissue are separated by gas, small expansions do not accumulate, thereby protecting against shattering. A small company, Arigos Biomedical, "has already recovered pig hearts from the 120 degrees below zero", although the definition of "recovered" is not clear. Pressures of 60 atm can help increase heat exchange rates. Gaseous oxygen perfusion/persufflation can enhance organ preservation relative to static cold storage or hypothermic machine perfusion, since the lower viscosity of gases, may help reach more regions of preserved organs and deliver more oxygen per gram tissue.

Freezable tissues

Generally, cryopreservation is easier for thin samples and suspended cells, because these can be cooled more quickly and so require lesser doses of

toxic Toxicity is the degree to which a chemical substance or a particular mixture of substances can damage an organism. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a subst ...

cryoprotectants. Therefore, cryopreservation of human

liver The liver is a major Organ (anatomy), organ only found in vertebrates which performs many essential biological functions such as detoxification of the organism, and the Protein biosynthesis, synthesis of proteins and biochemicals necessary for ...

s and

heart The heart is a muscular organ in most animals. This organ pumps blood through the blood vessels of the circulatory system. The pumped blood carries oxygen and nutrients to the body, while carrying metabolic waste such as carbon dioxide t ...

s for storage and

transplant Transplant or Transplantation may refer to: Sciences *Transplanting a plant from one location to another *Organ transplantation, moving an organ from one body to another *Transplant thought experiment, an experiment similar to Trolley problem *Tra ...

is still impractical. Nevertheless, suitable combinations of cryoprotectants and regimes of cooling and rinsing during warming often allow the successful cryopreservation of biological materials, particularly cell suspensions or thin tissue samples. Examples include: *

Semen Semen, also known as seminal fluid, is an organic bodily fluid created to contain spermatozoa. It is secreted by the gonads (sexual glands) and other sexual organs of male or hermaphroditic animals and can fertilize the female ovum. Semen i ...

semen cryopreservation Semen cryopreservation (commonly called sperm banking or sperm freezing) 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 tr ...

Blood Blood is a body fluid in the circulatory system of humans and other vertebrates that delivers necessary substances such as nutrients and oxygen to the cells, and transports metabolic waste products away from those same cells. Blood in the c ...

** Special cells for transfusion like platelets (Thrombosomes by Cellphire) **

Stem cell In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type o ...

s. It is optimal in high concentration of synthetic serum, stepwise equilibration and slow cooling. **

Genetic Material Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cla ...

Additionally, cryopreservation is used for gene therapy treatments e. g. for cancer patients suffering from leukemia or lymphoma. The genetic materials used for gene therapy have to be modified in vivo or ex vivo. In order to do that they need to be kept viable during transport and storage. With cryopreservation they are brought to ultra-low temperatures and are being thawed when needed. **

Umbilical cord blood Cord blood (umbilical cord blood) is blood that remains in the placenta and in the attached umbilical cord after childbirth. Cord blood is collected because it contains stem cells, which can be used to treat hematopoietic and genetic disorders su ...

in a

Cord blood bank A cord blood bank is a facility which stores umbilical cord blood for future use. Both private and public cord blood banks have developed in response to the potential for cord blood in treating diseases of the blood and immune systems. Public cord ...

* Tissue samples like

tumor A neoplasm () is a type of abnormal and excessive growth of tissue. The process that occurs to form or produce a neoplasm is called neoplasia. The growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists ...

s and histological cross sections * Eggs (

s) in

oocyte cryopreservation Human oocyte cryopreservation (egg freezing) is a procedure to preserve a woman's eggs (oocytes). This technique has been used to enable women to postpone pregnancy to a later date – whether for medical reasons (such as cancer treatment), or fo ...

Embryos An embryo is an initial stage of development of a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male sper ...

at cleavage stage (that are 2, 4, 8 or 16 cells) or at early blastocyst stage, in

embryo cryopreservation Cryopreservation of embryos is the process of Cryopreservation, preserving an embryo at sub-zero temperatures, generally at an embryogenesis stage corresponding to pre-implantation, that is, from fertilisation to the blastocyst stage. Indications ...

Ovarian tissue The ovary is an organ in the female reproductive system that produces an ovum. When released, this travels down the fallopian tube into the uterus, where it may become fertilized by a sperm. There is an ovary () found on each side of the body ...

in ovarian tissue cryopreservation *

Plant Plants are predominantly photosynthetic eukaryotes of the kingdom Plantae. Historically, the plant kingdom encompassed all living things that were not animals, and included algae and fungi; however, all current definitions of Plantae exclud ...

seeds or shoots tips or dormant buds are cryopreserved for

conservation Conservation is the preservation or efficient use of resources, or the conservation of various quantities under physical laws. Conservation may also refer to: Environment and natural resources * Nature conservation, the protection and manageme ...

purposes.

Embryos

Cryopreservation for embryos is used for embryo storage, e.g., when IVF has resulted in more embryos than is currently needed. One pregnancy and resulting healthy birth has been reported from an embryo stored for 27 years, after the successful pregnancy of an embryo from the same batch three years earlier. Many studies have evaluated the children born from frozen embryos, or “frosties”. The result has been uniformly positive with no increase in birth defects or development abnormalities. A study of more than 11,000 cryopreserved human embryos showed no significant effect of storage time on post-thaw survival for IVF or oocyte donation cycles, or for embryos frozen at the pronuclear or cleavage stages. Additionally, the duration of storage did not have any significant effect on clinical pregnancy, miscarriage, implantation, or live birth rate, whether from IVF or oocyte donation cycles. Rather, oocyte age, survival proportion, and number of transferred embryos are predictors of pregnancy outcome.

Ovarian tissue

Cryopreservation of ovarian tissue is of interest to women who want to preserve their reproductive function beyond the natural limit, or whose reproductive potential is threatened by cancer therapy, for example in hematologic malignancies or breast cancer. The procedure is to take a part of the ovary and perform slow freezing before storing it in liquid nitrogen whilst therapy is undertaken. Tissue can then be thawed and implanted near the fallopian, either orthotopic (on the natural location) or heterotopic (on the abdominal wall), where it starts to produce new eggs, allowing normal conception to occur. The ovarian tissue may also be transplanted into mice that are immunocompromised (

SCID mice The severe combined immunodeficiency (SCID) is a severe immunodeficiency genetic disorder that is characterized by the complete inability of the adaptive immune system to mount, coordinate, and sustain an appropriate immune response, usually due t ...

) to avoid graft rejection, and tissue can be harvested later when mature follicles have developed.

Oocytes

Human oocyte cryopreservation is a new technology in which a woman's eggs ( oocytes) are extracted, frozen and stored. Later, when she is ready to become pregnant, the eggs can be thawed, fertilized, and transferred to the uterus as

embryos An embryo is an initial stage of development of a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male sper ...

. Since 1999, when the birth of the first baby from an embryo-derived from vitrified-warmed woman's eggs was reported by Kuleshova and co-workers in the journal of Human Reproduction, this concept has been recognized and widespread. This breakthrough in achieving vitrification of a woman's oocytes made an important advance in our knowledge and practice of the IVF process, as the clinical pregnancy rate is four times higher after oocyte vitrification than after slow freezing. Oocyte vitrification is vital for preserving fertility in young oncology patients and for individuals undergoing IVF who object, for either religious or ethical reasons, to the practice of freezing embryos.

Semen

can be used successfully almost indefinitely after cryopreservation. The longest reported successful storage is 22 years. It can be used for

sperm donation Sperm donation is the provision by a man of his sperm with the intention that it be used in the artificial insemination or other 'fertility treatment' of a woman or women who are not his sexual partners in order that they may become pregnant by hi ...

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 Chemotherapy (often abbreviated to chemo and sometimes CTX or CTx) is a type of cancer treatment that uses one or more anti-cancer drugs (chemotherapeutic agents or alkylating agents) as part of a standardized chemotherapy regimen. Chemotherap ...

radiation therapy Radiation therapy or radiotherapy, often abbreviated RT, RTx, or XRT, is a therapy using ionizing radiation, generally provided as part of cancer treatment to control or kill malignant cells and normally delivered by a linear accelerator. Radia ...

or surgery.

Testicular tissue

Cryopreservation of immature testicular tissue is a developing method to avail reproduction to young boys who need to have gonadotoxic therapy. Animal data are promising since healthy offspring have been obtained after transplantation of frozen testicular cell suspensions or tissue pieces. However, none of the fertility restoration options from frozen tissue, i.e. cell suspension transplantation,

tissue grafting Grafting refers to a surgical procedure to move Tissue (biology), tissue from one site to another on the body, or from another creature, without bringing its own blood supply with it. Instead, a new blood supply grows in after it is placed. A s ...

and

in vitro maturation In vitro maturation (IVM) is the technique of letting the contents of ovarian follicles and the oocytes inside mature ''in vitro''. It can be offered to women with infertility problems, combined with In Vitro Fertilization (IVF), offering women pre ...

has proved efficient and safe in humans as yet.

Moss

Cryopreservation of whole

moss Mosses are small, non-vascular flowerless plants in the taxonomic division Bryophyta (, ) '' sensu stricto''. Bryophyta (''sensu lato'', Schimp. 1879) may also refer to the parent group bryophytes, which comprise liverworts, mosses, and hor ...

plant Plants are predominantly photosynthetic eukaryotes of the kingdom Plantae. Historically, the plant kingdom encompassed all living things that were not animals, and included algae and fungi; however, all current definitions of Plantae exclud ...

s, especially

Physcomitrella patens ''Physcomitrium patens'', (synonym: ''Physcomitrella patens'' ) the spreading earthmoss, is a moss (bryophyte) used as a model organism for studies on plant evolution, development, and physiology. Distribution and ecology ''Physcomitrella pat ...

, has been developed by

Ralf Reski Ralf Reski (born 18 November 1958 in Gelsenkirchen) is a German professor of plant biotechnology and former dean of the Faculty of Biology of the University of Freiburg. He is also affiliated to the French École supérieure de biotechnologie ...

and coworkers and is performed at the

International Moss Stock Center The International Moss Stock Center (IMSC) is a biorepository which is specialized in collecting, preserving and distributing moss plants of a high value of scientific research. The IMSC is located at the Faculty of Biology, Department of Plant ...

. This biobank collects, preserves, and distributes moss

mutant In biology, and especially in genetics, a mutant is an organism or a new genetic character arising or resulting from an instance of mutation, which is generally an alteration of the DNA sequence of the genome or chromosome of an organism. It ...

s and moss

ecotype In evolutionary ecology, an ecotype,Greek: ''οίκος'' = home and ''τύπος'' = type, coined by Göte Turesson in 1922 sometimes called ecospecies, describes a genetically distinct geographic variety, population, or race within a species, ...

Mesenchymal stromal cells (MSCs)

MSCs, when transfused immediately within a few hours post-thawing, may show reduced function or show decreased efficacy in treating diseases as compared to those MSCs which are in log phase of cell growth (fresh). As a result, cryopreserved MSCs should be brought back into the log phase of cell growth in ''in vitro'' culture before these are administered for clinical trials or experimental therapies. Re-culturing of MSCs will help in recovering from the shock the cells get during freezing and thawing. Various clinical trials on MSCs have failed which used cryopreserved products immediately post-thaw as compared to those clinical trials which used fresh MSCs.

Seed

Plant cryopreservation is becoming vital for its biodiversity value. Seeds are often considered as an important delivery system of genetic information. Cryopreservation of

recalcitrant seed Recalcitrant seeds are seeds that do not survive drying and freezing during ex-situ conservation. By and large, these seeds cannot resist the effects of drying or temperatures less than 10 °C (50 °F); thus, they cannot be stored for long periods ...

is the hardest due to intolerance to low temperature and low water content. However, plant vitrification solution can solve the problem and help recalcitrant seed (Nymphaea caerulea) cryopreserve.

Preservation of microbiology cultures

Bacteria and fungi can be kept short-term (months to about a year, depending) refrigerated, however, cell division and metabolism is not completely arrested and thus is not an optimal option for long-term storage (years) or to preserve cultures genetically or phenotypically, as cell divisions can lead to mutations or sub-culturing can cause phenotypic changes. A preferred option, species-dependent, is cryopreservation. Nematode worms are the only multicellular eukaryotes that have been shown to survive cryopreservation.

Fungi

Fungi, notably zygomycetes, ascomycetes, and higher basidiomycetes, regardless of sporulation, are able to be stored in liquid nitrogen or deep-frozen. Cryopreservation is a hallmark method for fungi that do not sporulate (otherwise other preservation methods for spores can be used at lower costs and ease), sporulate but have delicate spores (large or freeze-dry sensitive), are pathogenic (dangerous to keep metabolically active fungus) or are to be used for genetic stocks (ideally to have an identical composition as the original deposit). As with many other organisms, cryoprotectants like DMSO or glycerol (e.g. filamentous fungi 10% glycerol or yeast 20% glycerol) are used. Differences between choosing cryoprotectants are species (or class) dependent, but generally for fungi penetrating cryoprotectants like DMSO, glycerol or polyethylene glycol are most effective (other non-penetrating ones include sugars mannitol, sorbitol, dextran, etc.). Freeze-thaw repetition is not recommended as it can decrease viability. Back-up deep-freezers or liquid nitrogen storage sites are recommended. Multiple protocols for freezing are summarized below (each uses screw-cap polypropylene cryotubes):

Bacteria

Many common culturable laboratory strains are deep-frozen to preserve genetically and phenotypically stable, long-term stocks. Sub-culturing and prolonged refrigerated samples may lead to loss of plasmid(s) or mutations. Common final glycerol percentages are 15, 20, and 25. From a fresh culture plate, one single colony of interest is chosen and liquid culture is made. From the liquid culture, the medium is directly mixed with an equal amount of glycerol; the colony should be checked for any defects like mutations. All antibiotics should be washed from the culture before long-term storage. Methods vary, but mixing can be done gently by inversion or rapidly by vortex and cooling can vary by either placing the cryotube directly at −50 to −95 °C, shock-freezing in liquid nitrogen or gradually cooling and then storing at −80 °C or cooler (liquid nitrogen or liquid nitrogen vapor). Recovery of bacteria can also vary, namely, if beads are stored within the tube then the few beads can be used to plate or the frozen stock can be scraped with a loop and then plated, however, since only little stock is needed the entire tube should never be completely thawed and repeated freeze-thaw should be avoided. 100% recovery is not feasible regardless of methodology.

Freeze tolerance in animals

Worms

The microscopic soil-dwelling

nematode The nematodes ( or grc-gre, Νηματώδη; la, Nematoda) or roundworms constitute the phylum Nematoda (also called Nemathelminthes), with plant-Parasitism, parasitic nematodes also known as eelworms. They are a diverse animal phylum inhab ...

roundworms ''

Panagrolaimus detritophagus ''Panagrolaimus detritophagus'' is a terrestrial free-living nematode (roundworm). It has been reported in California, South America and Europe. It is the type species of the genus '' Panagrolaimus''. In 2018, it, along with another nematode spe ...

'' and '' Plectus parvus'' are the only eukaryotic organisms that have been proven to be viable after long-term cryopreservation to date. In this case, the preservation was natural rather than artificial, due to

permafrost Permafrost is ground that continuously remains below 0 °C (32 °F) for two or more years, located on land or under the ocean. Most common in the Northern Hemisphere, around 15% of the Northern Hemisphere or 11% of the global surface ...

Vertebrates

Several animal species, including fish, amphibians, and reptiles have been shown to tolerate freezing. At least four species of frogs ('' Pseudacris crucifer, Hyla versicolor, Pseudacris triseriata,

Lithobates sylvaticus ''Lithobates sylvaticus'' or ''Rana sylvatica'', commonly known as the wood frog, is a frog species that has a broad distribution over North America, extending from the Boreal forest of Canada, boreal forest of the north to the southern Appalach ...

'') and several species of turtles (''Terrapene carolina'', hatchling ''Chrysemys picta''), lizards, and snakes are freeze tolerant and have developed adaptations for surviving freezing. While some frogs hibernate underground or in water, body temperatures still drop to −5 to −7 °C, causing them to freeze. The wood frog (''Lithobates sylvaticus'') can withstand repeated freezing, during which about 65% of its extracellular fluid is converted to ice.