Cryopreservation

{{short description|Process to preserve biological matter}} {{pp-move}} {{lead too short|date=June 2012}} [[File:Cryopreservation USDA Gene Bank.jpg|thumb|upright=1.1|Cryogenically preserved samples being removed from a dewar of liquid nitrogen]]

'''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.<ref>{{cite book |doi=10.1007/978-1-4939-6921-0_5 |chapter=Cryopreservation: Vitrification and Controlled Rate Cooling |title=Stem Cell Banking |series=Methods in Molecular Biology |date=2017 |last1=Hunt |first1=Charles J. |volume=1590 |pages=41–77 |pmid=28353262 |isbn=978-1-4939-6919-7 }}</ref> At low temperatures (typically {{Convert|−80|C|F|abbr=unit|disp=b}} or {{Convert|-196|C|sigfig=3}} using liquid nitrogen) 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.

Plant materials that have been preserved through cyropreservation can theoretically remain alive for centuries. They are then properly removed and regenerated into healthy plants. As a result, cyropreservation has proven to be an effective method for conserving plant with unique genetic makeup and some species that produce recalcitrant seeds.<ref>{{Cite web |title=Cryopreservation |url=https://www.fao.org/plant-treaty/overview/partnerships/international-online-panels/international-expert-panel/en |access-date=2026-02-25 |website=PlantTreaty |language=en}}</ref>

Molecules, referred to as cryoprotective agents (CPAs), are added to reduce the osmotic shock and physical stresses cells undergo in the freezing process.<ref>{{cite journal |last1=Gurruchaga |first1=H |last2=Saenz del Burgo |first2=L |last3=Hernandez |first3=R.M |last4=Orive |first4=G |last5=Selden |first5=C |last6=Fuller |first6=B |last7=Ciriza |first7=J |last8=Pedraz |first8=J.L |title=Advances in the slow freezing cryopreservation of microencapsulated cells |journal=Journal of Controlled Release |date=July 2018 |volume=281 |pages=119–138 |doi=10.1016/j.jconrel.2018.05.016 |pmid=29782945 |url=https://discovery.ucl.ac.uk/id/eprint/10052556/ |url-access=subscription }}</ref> 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,<ref>{{Cite web |title=How do trees survive the winter? |url=https://www.nationalforests.org/blog/how-do-trees-survive-the-winter |access-date=2023-01-08 |website=www.nationalforests.org |language=en}}</ref><ref>{{cite book |doi=10.1016/B978-012088457-5/50021-6 |chapter=Impacts of Freezing on Long Distance Transport in Woody Plants |title=Vascular Transport in Plants |date=2005 |last1=Cavender-Bares |first1=Jeannine |pages=401–424 |isbn=978-0-12-088457-5 }}</ref> wood frogs,<ref>{{Cite web |date=2007-02-20 |title=Antifreeze-Like Blood Lets Frogs Freeze and Thaw With Winter's Whims |url=https://www.nationalgeographic.com/animals/article/frog-antifreeze-blood-winter-adaptation |archive-url=https://web.archive.org/web/20210302172652/https://www.nationalgeographic.com/animals/article/frog-antifreeze-blood-winter-adaptation |archive-date=March 2, 2021 |access-date=2023-01-08 |website=National Geographic }}</ref> and tardigrades.<ref>{{Cite web |last1=Mayer-Grenu |first1=rea |last2=Stuttgart |first2=University of |title=How tardigrades survive freezing temperatures |url=https://phys.org/news/2022-10-tardigrades-survive-temperatures.html |access-date=2023-01-08 |website=phys.org |language=en}}</ref>

The first human corpse to be frozen with the hope of future resurrection was James Bedford's, a few hours after his cancer-caused death in 1967.<ref name=":0" />

== Natural cryopreservation ==

Tardigrades, microscopic animals sometimes known as water bears, can survive freezing by replacing most of their internal water with a sugar called trehalose, preventing it from crystallization that otherwise damages cell membranes. Mixtures of solutes can achieve similar effects. Some solutes, including salts, have the disadvantage that they may be toxic at intense concentrations. Wood frogs can also 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 "cryoprotectants" to limit the amount of ice that forms and to reduce osmotic 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.{{citation needed|date=May 2014}}

'''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'', ''Pseudacris triseriata'', ''Hyla crucifer'', ''Hyla versicolor'', ''Hyla chrysoscelis''), one of salamanders (''Salamandrella keyserlingii''), one of snakes (''Thamnophis sirtalis'') and three of turtles (''Chrysemys picta'', ''Terrapene carolina'', ''Terrapene ornata'').<ref name=AJP1991 /> Snapping turtles ''Chelydra serpentina'' and wall lizards ''Podarcis muralis'' 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&nbsp;mmol/L when the frogs are cooled slowly.<ref name="AJP1991">{{cite journal |vauthors = Costanzo JP, Lee RE, Wright MF |title = Glucose loading prevents freezing injury in rapidly cooled wood frogs |journal = The American Journal of Physiology |volume = 261 | issue = 6 Pt 2 |pages = R1549–53 |date = December 1991 |pmid = 1750578 |doi = 10.1152/ajpregu.1991.261.6.R1549 |url = http://www.units.muohio.edu/cryolab/publications/documents/CostanzoLeeWright91AJP.pdf }}</ref>

== History == {{See also|Cryonics#History}} [[File:Petefészekszövet-csíkok fagyasztva tárolása.jpg|thumb|Tubes of biological samples being placed in liquid nitrogen]]

One early theoretician of cryopreservation was James Lovelock. In 1953, he suggested that damage to red blood cells during freezing was due to osmotic stress,<ref>{{cite journal | vauthors = Lovelock JE | title = The haemolysis of human red blood-cells by freezing and thawing | journal = Biochimica et Biophysica Acta | volume = 10 | issue = 3 | pages = 414–26 | date = March 1953 | pmid = 13058999 | doi = 10.1016/0006-3002(53)90273-X }}</ref> and that increasing the salt concentration in a dehydrating cell might damage it.<ref>{{cite book|editor-last1=Fuller|editor-first1=Barry J.|editor-last2=Lane |editor-first2=Nick|editor-last3=Benson|editor-first3=Erica E. | name-list-style = vanc |title=Life in the Frozen State|year=2004|publisher=CRC Press|url=https://books.google.com/books?id=PPven_q2xiQC&pg=PP7|page=7|isbn=978-0-203-64707-3}}</ref><ref>{{cite journal | vauthors = Mazur P | title = Cryobiology: the freezing of biological systems | journal = Science | volume = 168 | issue = 3934 | pages = 939–49 | date = May 1970 | pmid = 5462399 | doi = 10.1126/science.168.3934.939 | bibcode = 1970Sci...168..939M }}</ref> 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.<ref>{{cite magazine|title=The Cryobiological Case for Cryonics|magazine=Cryonics|page=27|date=March 1988|volume=9|number=3|id=Issue #92|publisher=Alcor Life Extension Foundation|url=https://www.alcor.org/cryonics/cryonics8803.pdf|access-date=2018-10-03|archive-date=2020-04-17|archive-url=https://web.archive.org/web/20200417212211/https://www.alcor.org/cryonics/cryonics8803.pdf}}</ref>

Cryopreservation was applied to human materials beginning in 1954 with three pregnancies resulting from the insemination of previously frozen sperm.<ref>{{cite news|title=Fatherhood After Death Has Now Been Proved Possible|newspaper=Cedar Rapids Gazette|date=April 9, 1954}}</ref> Fowl sperm was cryopreserved in 1957 by a team of scientists in the UK directed by Christopher Polge.<ref>{{cite journal | vauthors = Polge C | title = Low-temperature storage of mammalian spermatozoa | journal = Proceedings of the Royal Society of London. Series B, Biological Sciences | volume = 147 | issue = 929 | pages = 498–508 | date = December 1957 | pmid = 13494462 | doi = 10.1098/rspb.1957.0068 | bibcode = 1957RSPSB.147..498P }}</ref> <!--However, the rapid immersion of the samples in liquid nitrogen did not, for certain samples—such as some types of embryos, bone marrow and stem cells—produce the necessary viability to make them usable after thawing. Increased understanding of the mechanism of freezing injury to cells emphasized the importance of controlled or slow cooling to obtain maximum survival on thawing of the living cells. A controlled-rate cooling process, allowing biological samples to equilibrate to optimal physical parameters osmotically in a cryoprotectant (a form of anti-freeze) before cooling in a predetermined, controlled way proved necessary. The ability of cryoprotectants, in the early cases glycerol, to protect cells from freezing injury was discovered accidentally. Freezing injury has two aspects: direct damage from the ice crystals and secondary damage caused by the increase in the concentration of solutes as progressively more ice is formed.--> During 1963, Peter Mazur, at Oak Ridge National Laboratory 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&nbsp;°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.<ref>{{cite journal | vauthors = Mazur P | title = Studies on rapidly frozen suspensions of yeast cells by differential thermal analysis and conductometry | journal = Biophysical Journal | volume = 3 | issue = 4 | pages = 323–53 | date = July 1963 | pmid = 13934216 | pmc = 1366450 | doi = 10.1016/S0006-3495(63)86824-1 | url = | bibcode = 1963BpJ.....3..323M }}</ref>

On April 22, 1966, the first human cadaver was frozen—it had been embalmed for two months—by being placed in liquid nitrogen and stored at just above freezing. The cadaver was that of an elderly woman from Los Angeles, whose name is unknown, and was soon thawed out and buried by relatives. The first human corpse to be frozen with the hope of future resurrection was James Bedford's, a few hours after his cancer-caused death in 1967.<ref name=":0">{{cite web | title=Dear Dr. Bedford (and those who will care for you after I do) | publisher=Cryonics | date=July 1991 | url=http://www.alcor.org/Library/html/BedfordLetter.htm | access-date=2009-08-23 | archive-date=2012-09-16 | archive-url=https://web.archive.org/web/20120916103957/http://www.alcor.org/Library/html/BedfordLetter.htm }}</ref> Bedford's is the only cryonics corpse frozen before 1974 still frozen today.<ref>{{cite web|url=https://alcor.org/Library/html/suspensionfailures.html|title=Suspension Failures – Lessons from the Early Days|last=Perry|first=R. Michael|name-list-style=vanc|website=ALCOR: Life Extension Foundation|date=October 2014|access-date=August 29, 2018|archive-date=April 16, 2020|archive-url=https://web.archive.org/web/20200416155826/https://alcor.org/Library/html/suspensionfailures.html}}</ref>

== Risks ==

Phenomena which can cause damage to cells during cryopreservation mainly occur during the freezing stage, and include solution effects, extracellular 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.<ref>{{cite journal | doi=10.1152/ajpcell.1984.247.3.C125 | title=Freezing of living cells: Mechanisms and implications | date=1984 | last1=Mazur | first1=P. | journal=American Journal of Physiology. Cell Physiology | volume=247 | issue=3 | pages=C125–C142 | pmid=6383068 }}</ref>

; Solution effects: As ice crystals grow in freezing water, solutes 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 cells and ice 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 organisms and tissues can tolerate some extracellular ice, any appreciable intracellular ice is almost always fatal to cells.

{{anchor|methods}}

==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=== thumb|A tank of liquid nitrogen, used to supply a cryogenic freezer (for storing laboratory samples at a temperature of about {{Convert|−150|C|disp=or}})

''Controlled-rate and slow freezing'', also known as ''slow programmable freezing (SPF)'',<ref>{{cite journal | vauthors = Vutyavanich T, Piromlertamorn W, Nunta S | title = Rapid freezing versus slow programmable freezing of human spermatozoa | journal = Fertility and Sterility | volume = 93 | issue = 6 | pages = 1921–8 | date = April 2010 | pmid = 19243759 | doi = 10.1016/j.fertnstert.2008.04.076 | doi-access = free }}</ref> is a technique where cells are cooled to around -196&nbsp;°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 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 (IVF) births.<ref>{{cite web |url=http://www.bionews.org.uk/commentary.lasso?storyid=4055 |title=dead link |access-date=2020-07-26 }}{{dead link|date=May 2022|bot=medic}}{{cbignore|bot=medic}}</ref>

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,<ref>{{cite journal | vauthors = Deller RC, Vatish M, Mitchell DA, Gibson MI | title = Synthetic polymers enable non-vitreous cellular cryopreservation by reducing ice crystal growth during thawing | journal = Nature Communications | volume = 5 | article-number = 3244 | date = February 3, 2014 | pmid = 24488146 | doi = 10.1038/ncomms4244 | bibcode = 2014NatCo...5.3244D | doi-access = free }}</ref> biomaterials such as alginates, polyvinyl alcohol or chitosan can be used to impede ice crystal growth along with traditional small molecule cryoprotectants.<ref name="A Bayesian approach to optimizing c">{{cite journal |vauthors=Sambu S |date=June 25, 2015 |title=A Bayesian approach to optimizing cryopreservation protocols |journal=PeerJ |volume=3 |article-number=e1039 |doi=10.7717/peerj.1039 |pmc=4485240 |pmid=26131379 |doi-access=free }}</ref> That rate differs between cells of differing size and water permeability: a typical cooling rate of about 1&nbsp;°C/minute is appropriate for many mammalian cells after treatment with cryoprotectants such as glycerol or dimethyl sulfoxide (DMSO), but the rate is not a universal optimum. The 1&nbsp;°C / minute rate can be achieved by using devices such as a rate-controlled freezer or a benchtop portable freezing container.<ref>{{cite journal | vauthors = Thompson M, Nemits M, Ehrhardt R |title=Rate-controlled Cryopreservation and Thawing of Mammalian Cells |journal=Protocol Exchange |date=May 2011 |doi=10.1038/protex.2011.224 |url=https://www.nature.com/protocolexchange/protocols/2085|url-access=subscription |doi-access=free }}</ref>

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 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.<ref name="vitrification origination">{{cite journal|vauthors=Rall WF, Fahy GM|date=February 14–20, 1985|title=Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification|journal=Nature|volume=313|issue=6003|pages=573–5|bibcode=1985Natur.313..573R|doi=10.1038/313573a0|pmid=3969158 }}<!--|access-date=24 October 2012--></ref> As of 2000, researchers claim vitrification provides the benefits of cryopreservation without damage due to ice crystal formation.<ref name="cryonics 2000">{{cite news|url=https://www.alcor.org/cryonics/cryonics2000-4.pdf |title=Alcor: The Origin of Our Name |access-date=August 25, 2009 |date=Winter 2000 |publisher=Alcor Life Extension Foundation}}</ref> 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,<ref name="Kuleshova">{{cite journal | vauthors = Kuleshova LL, Wang XW, Wu YN, Zhou Y, Yu H | title = Vitrification of encapsulated hepatocytes with reduced cooling and warming rates | journal = Cryo Letters | volume = 25 | issue = 4 | pages = 241–54 | year = 2004 | pmid = 15375435 |url=https://www.ingentaconnect.com/content/cryo/cryo/2004/00000025/00000004/art00002 }}</ref> who also was the first scientist to achieve vitrification of oocytes, which resulted in live birth in 1999.<ref name="Kuleshova1999">{{cite journal | vauthors = Kuleshova L, Gianaroli L, Magli C, Ferraretti A, Trounson A | title = Birth following vitrification of a small number of human oocytes: case report | journal = Human Reproduction | volume = 14 | issue = 12 | pages = 3077–9 | date = December 1999 | pmid = 10601099 | doi = 10.1093/humrep/14.12.3077 | doi-access = free }}</ref> 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 crystallizing, the syrupy solution becomes an amorphous ice—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" 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.<ref>{{cite journal | vauthors = Bhat SN, Sharma A, Bhat SV | title = Vitrification and glass transition of water: insights from spin probe ESR | journal = Physical Review Letters | volume = 95 | issue = 23 | article-number = 235702 | date = December 2005 | pmid = 16384318 | doi = 10.1103/PhysRevLett.95.235702 | arxiv = cond-mat/0409440 | bibcode = 2005PhRvL..95w5702B }}</ref>

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 company to vitrify a rabbit kidney to −135&nbsp;°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.<ref>{{cite journal | vauthors = Fahy GM, Wowk B, Pagotan R, Chang A, Phan J, Thomson B, Phan L | title = Physical and biological aspects of renal vitrification | journal = Organogenesis | volume = 5 | issue = 3 | pages = 167–75 | date = July 2009 | pmid = 20046680 | pmc = 2781097 | doi = 10.4161/org.5.3.9974 }}</ref> In 2000, FM-2030 became the first person to be successfully vitrified posthumously.<ref>{{cite journal |url=https://www.alcor.org/cryonics/cryonics2000-4.pdf |title=A Tribute to FM-2030 |access-date=2009-08-25 |last=Chamberlain |first=Fred |journal=Cryonics |volume=21 |issue=4 |page=11 |date=Winter 2000 |archive-date=November 19, 2010 |archive-url=https://web.archive.org/web/20101119134918/http://alcor.org/cryonics/cryonics2000-4.pdf |url-status=live }}</ref>

===Persufflation=== Blood can be replaced with inert noble gases and/or metabolically vital gases like oxygen, 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.<ref>{{cite news |last1=Geddes |first1=Linda |title=Heart of glass could be key to banking organs |url=https://www.newscientist.com/article/mg21929343-100-heart-of-glass-could-be-key-to-banking-organs/ |work=New Scientist |date=11 September 2013 }}</ref> A small company, Arigos Biomedical, "has already recovered pig hearts from the 120 degrees below zero",<ref>{{cite journal | journal=BOSS Magazine | first = Matthew | last = Flynn | name-list-style = vanc | date=Oct 10, 2018 | title= Heart of Ice |url=https://thebossmagazine.com/freezing-and-thawing-organs/}}</ref> although the definition of "recovered" is not clear. Pressures of 60 atm can help increase heat exchange rates.<ref>{{cite patent|country=US|number=9314015|title=Method and apparatus for prevention of thermo-mechanical fracturing in vitrified tissue using rapid cooling and warming by persufflation|assign1=Arigos Biomedical Inc.|inventor1-last=Van Sickle|inventor1-first=Stephen|inventor2-last=Jones|inventor2-first=Tanya|pubdate=2016-04-19}}</ref> 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.<ref name="pmid22301419">{{cite journal | vauthors = Suszynski TM, Rizzari MD, Scott WE, Tempelman LA, Taylor MJ, Papas KK | title = Persufflation (or gaseous oxygen perfusion) as a method of organ preservation | journal = Cryobiology | volume = 64 | issue = 3 | pages = 125–43 | date = June 2012 | pmid = 22301419 | pmc = 3519283 | doi = 10.1016/j.cryobiol.2012.01.007 }}</ref>

== Freezable tissues and organs ==

Generally, cryopreservation is easier for thin samples and suspended cells, because these can be cooled more quickly and so require lesser doses of toxic cryoprotectants. Therefore, '''tissue cryopreservation''' of human livers and hearts ('''organ cryopreservation''') for storage and transplant is still impractical or experimental.<ref>{{cite journal|title=Organ preservation: current limitations and optimization approaches|first1=Qiulin|last1=Ran|first2=Jiayi|last2=Zhang|first3=Jisheng|last3=Zhong|first4=Ji|last4=Lin|first5=Shuai|last5=Zhang|first6=Guang|last6=Li|first7=Bin|last7=You|journal=Frontiers in Medicine|date=26 March 2025 |volume=12 |article-number=1566080 |doi=10.3389/fmed.2025.1566080 |pmid=40206471 |pmc=11980443 |doi-access=free }}</ref><ref name=PubMed02>{{cite journal|title=Current State and Challenges of Tissue and Organ Cryopreservation in Biobanking|journal=International Journal of Molecular Sciences|first1=Irina|last1=Khaydukova|first2=Valeria|last2=Ivannikova|first3=Dmitry|last3=Zhidkov|first4=Nikita|last4=Belikov|first5=Maria|last5=Peshkova|first6=Peter|last6=Timashev|first7=Dmitry|last7=Tsiganov|first8=Aleksandr|last8=Pushkarev |date=2024 |volume=25 |issue=20 |article-number=11124 |doi=10.3390/ijms252011124 |doi-access=free |pmid=39456905 |pmc=11508709 }}</ref><ref name=PubMed03>{{cite journal|title=The cryopreservation of composite tissues|journal=Organogenesis|first1=Joseph|last1=Bakhach |date=2009 |volume=5 |issue=3 |pages=119–126 |doi=10.4161/org.5.3.9583 |pmid=20046674 |pmc=2781091 }}</ref> Most organs are usually preserved at a temperature of just above 0°C, which allows them to be stored for a few hours to a few days. Certain organs may be preserved at temperatures between -20°C to -50°C, enabling storage for a few weeks to a few months. In 2023, researchers successfully cryopreserved rat kidneys at -196°C using liquid nitrogen for 100 days.<ref name=PubMed04>{{cite journal|title=Vitrification and nanowarming enable long-term organ cryopreservation and life-sustaining kidney transplantation in a rat model|journal=Nature Communications|first1=Zonghu|last1=Han|first2=Joseph|last2=Rao|first3=Lakshya|last3=Gangwar|first4=Bat|last4=Namsrai|first5=Jacqueline|last5=Allen|first6=Michael|last6=Etheridge|first7=Susan|last7=Wolf|first8=Timothy|last8=Pruett|first9=John|last9=Bischof|first10=Erik|last10=Finger |date=2023 |volume=14 |issue=1 |article-number=3407 |doi=10.1038/s41467-023-38824-8 |pmid=37296144 |pmc=10256770 |bibcode=2023NatCo..14.3407H }}</ref>

With 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. At -196 °C using liquid nitrogen, tissues and organs can be preserved for a long period, often more than a decade.<ref name=PubMed01>{{cite journal|title=Cryopreservation of tissues and organs: present, bottlenecks, and future|journal= Frontiers in Veterinary Science|first1=Jiangming|last1=Chen|first2=Xiangjian|last2=Liu|first3=Yuying|last3=Hu|first4= Xiaoxiao|last4=Chen|first5=Songwen|last5=Tan |date=2023 |volume=10 |article-number=1201794 |doi=10.3389/fvets.2023.1201794 |doi-access=free |pmid=37303729 |pmc=10248239 }}</ref><ref name=PubMed02/><ref name=PubMed03/>

Examples include: * Semen in semen cryopreservation * Blood ** Special cells for transfusion like platelets (Thrombosomes by Cellphire) ** Stem cells. It is optimal in high concentration of synthetic serum, stepwise equilibration and slow cooling.<ref>{{cite journal | vauthors = Lee JY, Lee JE, Kim DK, Yoon TK, Chung HM, Lee DR | title = High concentration of synthetic serum, stepwise equilibration and slow cooling as an efficient technique for large-scale cryopreservation of human embryonic stem cells | journal = Fertility and Sterility | volume = 93 | issue = 3 | pages = 976–85 | date = February 2010 | pmid = 19022437 | doi = 10.1016/j.fertnstert.2008.10.017 | doi-access = free }}</ref> ** Genetic Material 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 thawed when needed.<ref>{{cite web |last1=Fischer |first1=Barbara |title=Cryopreservation: What you need to know about cryogenic freezing |url=https://www.susupport.com/cryopreservation-process |website=www.susupport.com |access-date=3 August 2022}}</ref> ** Umbilical cord blood in a Cord blood bank * Tissue samples like tumors and histological cross sections * Eggs (oocytes) in oocyte cryopreservation * Embryos at cleavage stage (that are 2, 4, 8 or 16 cells) or at early blastocyst stage, in embryo cryopreservation * Ovarian tissue in ovarian tissue cryopreservation * Plant seeds, callus, shoots tips or dormant buds are cryopreserved for conservation purposes.<ref>{{cite journal | vauthors = Panis B, Nagel M, Van den houwe I | title = Challenges and prospects for the conservation of crop genetic resources in field genebanks, in in vitro collections and/or in liquid nitrogen | journal = Plants | volume = 9 | issue = 12 | page = 1634 | date = November 2020 | doi = 10.3390/plants9121634 | pmid = 33255385 | pmc = 7761154 | doi-access = free | bibcode = 2020Plnts...9.1634P }}</ref><ref>{{cite journal | vauthors = Malek Zadeh S | title = ICryopreservation of the axial meristem of Crocus sativus L. | journal = Cryobiology | volume = 59 | page = 412 | year = 2009 | issue = 3 | doi = 10.1016/j.cryobiol.2009.10.163}}</ref><ref>{{cite journal |last1=Mikuła |first1=Anna |last2=Chmielarz |first2=Paweł |last3=Hazubska-Przybył |first3=Teresa |last4=Kulus |first4=Dariusz |last5=Maślanka |first5=Małgorzata |last6=Pawłowska |first6=Bożena |last7=Zimnoch-Guzowska |first7=Ewa |title=Cryopreservation of Plant Tissues in Poland: Research Contributions, Current Status, and Applications |journal=Acta Societatis Botanicorum Poloniae |date=14 December 2022 |volume=91 |article-number=9132 |doi=10.5586/asbp.9132 |doi-access=free |bibcode=2022AcSBP..91.9132M }}</ref>

===Embryos=== <!--Embryo storage redirects here--> {{Main|Embryo cryopreservation}} 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.<ref>{{cite news |last1=Cramer |first1=Maria |title=Girl Is Born in Tennessee From Embryo Frozen for 27 Years |url=https://www.nytimes.com/2020/12/03/science/tennessee-embryo-donate.html |work=The New York Times |date=3 December 2020 }}</ref> 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.<ref>{{cite web|url=http://www.givf.com/fertility/embryofreezing.cfm |archive-url=https://archive.today/20121206043620/http://www.givf.com/fertility/embryofreezing.shtml |archive-date=December 6, 2012 |title=Genetics & IVF Institute |publisher=Givf.com |access-date=July 27, 2009 }}</ref> 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.<ref name=riggs>{{cite journal | vauthors = Riggs R, Mayer J, Dowling-Lacey D, Chi TF, Jones E, Oehninger S | title = Does storage time influence postthaw survival and pregnancy outcome? An analysis of 11,768 cryopreserved human embryos | journal = Fertility and Sterility | volume = 93 | issue = 1 | pages = 109–15 | date = January 2010 | pmid = 19027110 | doi = 10.1016/j.fertnstert.2008.09.084 | doi-access = free }}</ref> 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.<ref name=riggs/> Rather, oocyte age, survival proportion, and number of transferred embryos are predictors of pregnancy outcome.<ref name=riggs/>

=== Ovarian tissue === {{Main|Ovarian tissue cryopreservation}} 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,<ref>{{cite journal | vauthors = Isachenko V, Lapidus I, Isachenko E, Krivokharchenko A, Kreienberg R, Woriedh M, Bader M, Weiss JM | display-authors = 6 | title = Human ovarian tissue vitrification versus conventional freezing: morphological, endocrinological, and molecular biological evaluation | journal = Reproduction | volume = 138 | issue = 2 | pages = 319–27 | date = August 2009 | pmid = 19439559 | doi = 10.1530/REP-09-0039 | doi-access = free }}</ref> for example in hematologic malignancies or breast cancer.<ref name=Oktay>{{cite journal | vauthors = Oktay K, Oktem O | title = Ovarian cryopreservation and transplantation for fertility preservation for medical indications: report of an ongoing experience | journal = Fertility and Sterility | volume = 93 | issue = 3 | pages = 762–8 | date = February 2010 | pmid = 19013568 | doi = 10.1016/j.fertnstert.2008.10.006 | doi-access = free }}</ref> 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),<ref name=Oktay/> where it starts to produce new eggs, allowing normal conception to occur.<ref>{{cite journal |last1=Donnez |first1=J |last2=Dolmans |first2=Mm |last3=Demylle |first3=D |last4=Jadoul |first4=P |last5=Pirard |first5=C |last6=Squifflet |first6=J |last7=Martinez-Madrid |first7=B |last8=Van Langendonckt |first8=A |title=Livebirth after orthotopic transplantation of cryopreserved ovarian tissue |journal=The Lancet |date=October 2004 |volume=364 |issue=9443 |pages=1405–1410 |doi=10.1016/S0140-6736(04)17222-X |pmid=15488215 }}</ref> The ovarian tissue may also be transplanted into mice that are immunocompromised (SCID mice) to avoid graft rejection, and tissue can be harvested later when mature follicles have developed.<ref>{{cite journal | vauthors = Lan C, Xiao W, Xiao-Hui D, Chun-Yan H, Hong-Ling Y | title = Tissue culture before transplantation of frozen-thawed human fetal ovarian tissue into immunodeficient mice | journal = Fertility and Sterility | volume = 93 | issue = 3 | pages = 913–9 | date = February 2010 | pmid = 19108826 | doi = 10.1016/j.fertnstert.2008.10.020 | doi-access = free }}</ref>

===Oocytes=== {{Main|Oocyte cryopreservation}} 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. 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,<ref name=Kuleshova/> 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.<ref>{{cite journal | vauthors = Glujovsky D, Riestra B, Sueldo C, Fiszbajn G, Repping S, Nodar F, Papier S, Ciapponi A | year = 2014| title = Vitrification versus slow freezing for women undergoing oocyte cryopreservation | journal = Cochrane Database of Systematic Reviews | issue = 9| article-number = CD010047 | doi = 10.1002/14651858.CD010047.pub2 | pmid = 25192224 | pmc = 11246547 }}</ref> 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=== {{Main|Semen cryopreservation}} Semen can be used successfully almost indefinitely after cryopreservation. The longest reported successful storage is 22 years.<ref>[http://www.planer.com/PlanerComWebsite.nsf/7dc62b5d9e44dd40802575460048fc4e/df90c37237a74df88025757d005b0057?OpenDocument Planer NEWS and Press Releases > Child born after 22-year semen storage using Planer controlled rate freezer] {{Webarchive|url=https://archive.today/20120908021925/http://www.planer.com/PlanerComWebsite.nsf/7dc62b5d9e44dd40802575460048fc4e/df90c37237a74df88025757d005b0057?OpenDocument |date=2012-09-08 }} 14/10/2004</ref> 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.

===Testicular tissue=== {{Main|Cryopreservation of 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 and in vitro maturation has proved efficient and safe in humans as yet.<ref>{{cite journal | vauthors = Wyns C, Curaba M, Vanabelle B, Van Langendonckt A, Donnez J | title = Options for fertility preservation in prepubertal boys | journal = Human Reproduction Update | volume = 16 | issue = 3 | pages = 312–28 | date = 2010 | pmid = 20047952 | doi = 10.1093/humupd/dmp054 | doi-access = free }}</ref>

===Moss=== [[Image:Ecotypes of Physcomitrella patens.JPG|thumb|200px|Four different ecotypes of ''Physcomitrella patens'' stored at the International Moss Stock Center]] Cryopreservation of whole moss plants, especially Physcomitrella patens, has been developed by Ralf Reski and coworkers<ref>{{cite journal | vauthors = Schulte J, Reski R | title = High throughput cryopreservation of 140,000 Physcomitrella patens mutants | journal = Plant Biology | volume = 6 | issue = 2 | pages = 119–27 | year = 2004 | pmid = 15045662 | doi = 10.1055/s-2004-817796 | publisher = Plant Biotechnology, Freiburg University, Freiburg, Germany | bibcode = 2004PlBio...6..119S }}</ref> and is performed at the International Moss Stock Center. This biobank collects, preserves, and distributes moss mutants and moss ecotypes.<ref>{{cite press release |title=Mosses, deep-frozen |url=https://www.sciencedaily.com/releases/2010/02/100224134325.htm |work=ScienceDaily |publisher=Albert-Ludwigs-Universität Freiburg |date=6 March 2010 }}</ref>

===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.<ref>{{cite journal | vauthors = François M, Copland IB, Yuan S, Romieu-Mourez R, Waller EK, Galipeau J | title = Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-γ licensing | journal = Cytotherapy | volume = 14 | issue = 2 | pages = 147–52 | date = February 2012 | pmid = 22029655 | pmc = 3279133 | doi = 10.3109/14653249.2011.623691 }}</ref>

=== 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 is the hardest due to intolerance to low temperature and low water content.<ref>{{cite journal |last1=Roque-Borda |first1=Cesar Augusto |last2=Kulus |first2=Dariusz |last3=Vacaro de Souza |first3=Angela |last4=Kaviani |first4=Behzad |last5=Vicente |first5=Eduardo Festozo |title=Cryopreservation of Agronomic Plant Germplasm Using Vitrification-Based Methods: An Overview of Selected Case Studies |journal=International Journal of Molecular Sciences |date=7 June 2021 |volume=22 |issue=11 |page=6157 |doi=10.3390/ijms22116157 |doi-access=free |pmid=34200414 |pmc=8201202 }}</ref> However, plant vitrification solution can solve the problem and help recalcitrant seed (Nymphaea caerulea) cryopreserve.<ref>{{cite thesis |author1=李崇豪 |date=2016 |title=埃及藍睡蓮種子的冷凍保存 — 使用添加穀胱甘肽的植物抗凍配方 |trans-title=Cryopreservation of seeds of blue waterlily (Nymphaea caerulea) using glutathione adding plant vitrification solution, PVS+ |oclc=1009363362 |hdl=11296/477356 |language=zh-Hant-TW }}{{pn|date=December 2024}}</ref>

==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.<ref>{{cite news |last1=Weisberger |first1=Mindy |title=Worms Frozen for 42,000 Years in Siberian Permafrost Wriggle to Life |url=https://www.livescience.com/63187-siberian-permafrost-worms-revive.html |work=Live Science |date=27 July 2018 }}</ref><ref>{{cite journal | vauthors = Shatilovich AV, Tchesunov AV, Neretina TV, Grabarnik IP, Gubin SV, Vishnivetskaya TA, Onstott TC, Rivkina EM | title = Viable Nematodes from Late Pleistocene Permafrost of the Kolyma River Lowland | journal = Doklady Biological Sciences | volume = 480 | issue = 1 | pages = 100–102 | date = May 2018 | pmid = 30009350 | doi = 10.1134/S0012496618030079 }}</ref>

===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):<ref>{{cite book |doi=10.1016/B978-012509551-8/50006-4 |chapter=Preservation and Distribution of Fungal Cultures |title=Biodiversity of Fungi |date=2004 |last1=Nakasone |first1=Karen K. |last2=Peterson |first2=Stephen W. |last3=Jong |first3=Shung-Chang |pages=37–47 |isbn=978-0-12-509551-8 }}</ref>

===Bacteria=== Many common culturable laboratory strains are deep-frozen to preserve genetically and phenotypically stable, long-term stocks.<ref name="Vitt, Laurie J">{{cite book |doi=10.1016/C2010-0-67152-5 |title=Herpetology |date=2014 |isbn=978-0-12-386919-7 |first1=Laurie J. |last1=Vitt |first2=Janalee P. |last2=Caldwell }}{{pn|date=December 2024}}</ref> 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&nbsp;°C, shock-freezing in liquid nitrogen or gradually cooling and then storing at −80&nbsp;°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.<ref>Freeze-Drying and Cryopreservation of Bacteria</ref><ref>{{cite web|url=https://www.addgene.org/plasmid_protocols/create_glycerol_stock/|title=Addgene: Protocol - How to Create a Bacterial Glycerol Stock|website=Addgene.org|access-date=9 September 2015}}</ref><ref>{{Cite web |url=https://www.qiagen.com/knowledge-and-support/spotlight/plasmid-resource-center/growth%20of%20bacterial%20cultures/ |title=Growth of Bacterial Cultures |access-date=2014-05-15 |archive-url=https://web.archive.org/web/20130907031938/http://www.qiagen.com/Knowledge-and-Support/Spotlight/Plasmid-Resource-Center/Growth%20of%20bacterial%20cultures |archive-date=2013-09-07 }}</ref>

== Freeze tolerance in animals ==

=== Worms === The microscopic soil-dwelling nematode roundworms ''Panagrolaimus detritophagus'' and ''Plectus parvus'' are the only eukaryotic organisms that have been proven to be viable after cryopreservation for many years (30,000 to 40,000 years). In this case, the preservation was natural rather than artificial, due to permafrost. They came alive when warmed up.

=== 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'') 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&nbsp;°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.<ref name="Vitt, Laurie J"/>

Rats have been reanimated from near-0 (but not frozen) temperatures.<ref>{{cite journal|pmc=1365902|journal=Journal of Physiology|year=1955|volume=128|pp=541&ndash;546|title=Reanimation of rats from body temperatures between 0 and 1&nbsp;°C by microwave diathermy|first1=R. K.|last1=Andjus|first2=J. E.|last2=Lovelock|orig-date=3 Dec 1954}}</ref>

== See also == * Aldehyde-stabilized cryopreservation * Cells Alive System freezers * Cryobiology * Cryogenic processor * Cryogenics * Cryopreservation of testicular tissue * Tissue cryopreservation * Organ cryopreservation * Cryostasis (clathrate hydrates) * Directional freezing * Ex-situ conservation * Frozen zoo * Plant cryopreservation—Cryoconservation of plant genetic resources

== References == {{Reflist}}

== Further reading == {{refbegin|}} * {{cite book|veditors=Engelmann F, Dulloo ME, Astorga C, Dussert S, Anthony F|date=2007|title=Conserving coffee genetic resources|publisher=Bioversity International, CATIE, IRD|url=http://www.bioversityinternational.org/Publications/pubfile.asp?ID_PUB=1244|page=61|access-date=2007-12-12|archive-date=2007-12-04|archive-url=https://web.archive.org/web/20071204082021/http://www.bioversityinternational.org/Publications/pubfile.asp?ID_PUB=1244}} * {{cite book|vauthors=Panis B|title=Cryopreservation of Musa germplasm: 2nd Edition|publisher=Bioversity International|year=2009|isbn=978-2-910810-86-3|url=https://www.bioversityinternational.org/fileadmin/_migrated/uploads/tx_news/Cryopreservation_of_Musa_germplasm__2nd_edition_1383.pdf|location=Montpellier, France|page=51}} * {{cite web|author=ReproTech Limited|date=2012|title=Fertility Preservation|publisher=ReproTech Limited|url=http://reprotech.com/cryostorage/fertility-preservation/about-fertility-preservation.html|archive-url=https://web.archive.org/web/20120904082833/http://reprotech.com/cryostorage/fertility-preservation/about-fertility-preservation.html|archive-date=2012-09-04}} * {{cite book|vauthors=Nakasone KK, Peterson SW, Jong SC|chapter=Preservation and distribution of fungal cultures.|title=Biodiversity of fungi: inventory and monitoring methods.|url=https://archive.org/details/biodiversityfung00fost|url-access=limited|location=Amsterdam|publisher=Elsevier Academic Press|date=2004|pages=[https://archive.org/details/biodiversityfung00fost/page/n44 37]–47|isbn=978-0-12-509551-8}} * {{cite book|vauthors=Perry SF|title=Cryopreservation and Freeze-Drying Protocols|chapter=Freeze-drying and cryopreservation of bacteria|series=Methods in Molecular Biology|location=Clifton, N.J.|volume=38|pages=21–30|date=1995|pmid=7647859|doi=10.1385/0-89603-296-5:21|isbn=0-89603-296-5}} {{refend}}

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Category:Cryopreservation Category:Preservation methods