Cryopreservation

Cryo-preservation or cryo-conservation is a process where organelles, cells, tissues, extracellular matrix, organs, or any other biological constructs susceptible to damage caused by unregulated chemical kinetics are preserved by cooling to very low temperatures[1] (typically −80 °C using solid carbon dioxide or −196 °C using liquid nitrogen). At low enough temperatures, any enzymatic 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 cryoprotectants. New methods are constantly being investigated due to the inherent toxicity of many cryoprotectants.[2] By default it should be considered that cryopreservation alters or compromises the structure and function of cells unless it is proven otherwise for a particular cell population. Cryoconservation of animal genetic resources is the process in which animal genetic material is collected and stored with the intention of conservation of the breed.

Petefészekszövet-csíkok fagyasztva tárolása
Tubes of biological samples being placed in liquid nitrogen.
Cryopreservation USDA Gene Bank
Cryogenically preserved samples being removed from a liquid nitrogen dewar.

Natural cryopreservation

Water-bears (Tardigrada), microscopic multicellular organisms, can survive freezing by replacing most of their internal water with the sugar 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. 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 "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.

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 (Hynobius keyserlingi), one of snakes (Thamnophis sirtalis) and three of turtles (Chrysemys picta, Terrapene carolina, Terrapene ornata).[3] 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 mmol/l when the frogs are cooled slowly.[3]

History

One of the most important early theoreticians of cryopreservation was James Lovelock. In 1953, he suggested that damage to red blood cells during freezing was due to osmotic stress,[4] and that increasing the salt concentration in a dehydrating cell might damage it.[5][6] 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.[7] This work led other scientists to attempt the short-term freezing of rats by 1955, which were fully active 4 to 7 days after being revived.[8]

Cryopreservation was applied to humans beginning in 1954 with three pregnancies resulting from the insemination of previously frozen sperm.[9] Fowl sperm was cryopreserved in 1957 by a team of scientists in the UK directed by Christopher Polge.[10] 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 °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.[11]

The first human body to be frozen with the hope of future revival was James Bedford's, a few hours after his cancer-caused death in 1967.[12] Bedford is the only cryonics patient frozen before 1974 still preserved today.[13]

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 (Tg) of polyol's water solutions, around −136 °C (137 K; −213 °F), seem to be accepted as the range where biological activity very substantially slows, and −196 °C (77 K; −321 °F), the boiling point of liquid nitrogen, is the preferred temperature for storing important specimens. While refrigerators, 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 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. However, estimates based on the accumulation of radiation-induced DNA damage during cryonic storage have suggested a maximum storage period of 1000 years.[14]

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.

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

Liquid nitrogen tank
A tank of liquid nitrogen, used to supply a cryogenic freezer (for storing laboratory samples at a temperature of about −150 °C)

Controlled-rate and slow freezing, also known as slow programmable freezing (SPF),[15] is a set of well established techniques developed during the early 1970s which enabled the first human embryo frozen birth Zoe Leyland during 1984. Since then, machines that freeze biological samples using programmable sequences, or controlled rates, have been used all over the world 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, embryo, 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 fertilisation (IVF) births.[16]

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 crystal growth and recrystallization,[17] biomaterials such as alginates, polyvinyl alcohol or chitosan can be used to impede ice crystal growth along with traditional small molecule cryoprotectants.[18] 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 or dimethyl sulfoxide, 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.[19]

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

Researchers Greg Fahy and William F. Rall helped to introduce vitrification to reproductive cryopreservation in the mid-1980s.[20] As of 2000, researchers claim vitrification provides the benefits of cryopreservation without damage due to ice crystal formation.[21] 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,[22] who also was the first scientist to achieve vitrification of oocytes, which resulted in live birth in 1999.[23] For clinical cryopreservation, vitrification usually requires the addition of cryoprotectants prior to cooling. The cryoprotectants act like antifreeze: they decrease the freezing temperature. They also increase the viscosity. 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.[24]

Two conditions usually required to allow vitrification are an increase of the viscosity and a decrease of 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 freezing temperature inside the cell. Sugars do not readily permeate through the membrane. Those solutes that do, such as dimethyl sulfoxide, 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 Twenty-First Century Medicine company to vitrify a rabbit kidney 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.[25]

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.[26] A small company, Arigos Biomedical, "has already recovered pig hearts from the 120 degrees below zero",[27] although the definition of "recovered" is not clear. Pressures of 60 atm can help increase heat exchange rates.[28] 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.[29]

Freezable tissues

Generally, cryopreservation is easier for thin samples and small clumps of individual cells, because these can be cooled more quickly and so require lesser doses of toxic cryoprotectants. Therefore, cryopreservation of human livers and hearts for storage and transplant 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:

Additionally, efforts are underway to preserve humans cryogenically, known as cryonics. For such efforts either the brain within the head or the entire body may experience the above process. Cryonics is in a different category from the aforementioned examples, however: while countless cryopreserved cells, vaccines, tissue and other biological samples have been thawed and used successfully, this has not yet been the case at all for cryopreserved brains or bodies. At issue are the criteria for defining "success".

Proponents of cryonics claim that cryopreservation using present technology, particularly vitrification of the brain, may be sufficient to preserve people in an "information theoretic" sense so that they could be revived and made whole by hypothetical vastly advanced future technology. Not only is there no guarantee of its success, many people argue that human cryopreservation is unethical. According to certain views of the mind body problem, some philosophers believe that the mind, which contains thoughts, memories, and personality, is separate from the brain. When someone dies, their mind leaves the body. If a cryopreserved patient gets successfully resuscitated, no one knows if they would be the same person that they once were or if they would be an empty shell of the memory of who they once were.

Right now scientists are trying to see if transplanting cryopreserved human organs for transplantation is viable, if so this would be a major step forward for the possibility of reviving a cryopreserved human.[31]

Embryos

Cryopreservation for embryos is used for embryo storage, e.g., when in vitro fertilization (IVF) has resulted in more embryos than is currently needed.

Pregnancies have been reported from embryos stored for 16 years.[32] 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.[33] 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.[34] 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.[34] Rather, oocyte age, survival proportion, and number of transferred embryos are predictors of pregnancy outcome.[34]

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,[35] for example in hematologic malignancies or breast cancer.[36] 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),[36] where it starts to produce new eggs, allowing normal conception to occur.[37] 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.[38]

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. 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,[22] this concept has been recognized and widespread. This break-through in achieving vitrification of woman’s oocytes made an important advance in our knowledge and practice of the IVF process, as clinical pregnancy rate is four times higher after oocyte vitrification than after slow freezing.[39] Oocyte vitrification is vital for preservation fertility in young oncology patients and for individuals undergoing IVF who object, either for religious or ethical reasons, to the practice of freezing embryos.

Semen

Semen can be used successfully almost indefinitely after cryopreservation. The longest reported successful storage is 22 years.[40] 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

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 (IVM) has proved efficient and safe in humans as yet.[41]

Moss

Ecotypes of Physcomitrella patens
Four different ecotypes of Physcomitrella patens stored at the IMSC.

Cryopreservation of whole moss plants, especially Physcomitrella patens, has been developed by Ralf Reski and coworkers[42] and is performed at the International Moss Stock Center. This biobank collects, preserves, and distributes moss mutants and moss ecotypes.[43]

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 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.[44]

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. [45]Shatilovich AV, Tchesunov AV, Neretina TV, Grabarnik IP, Gubin SV, Vishnivetskaya TA, Onstott TC, Rivkina EM (May 2018). "Viable Nematodes from Late Pleistocene Permafrost of the Kolyma River Lowland". Doklady Biological Sciences : Proceedings of the Academy of Sciences of the USSR, Biological Sciences Sections. 480 (1): 100–102. doi:10.1134/S0012496618030079. PMID 30009350.

Fungi

Fungi, notably zygomycetes, ascomycetes and higher basidiomycetes, regardless of sporulation, are able to be stored in liquid nitrogen or deep-frozen. Crypreservation 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 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):[46]

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 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.[47][48][49]

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 long-term cryopreservation to date. In this case, the preservation was natural rather than artificial, due to permafrost.

See also

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  49. ^ "Archived copy". Archived from the original on 2013-09-07. Retrieved 2014-05-15.CS1 maint: Archived copy as title (link)

Further reading

  • Engelmann F, Dulloo ME, Astorga C, Dussert S, Anthony F, eds. (2007). Conserving coffee genetic resources. Bioversity International, CATIE, IRD. p. 61.
  • Panis B, Tien Thinh N (2001). Cryopreservation of Musa germplasm. INIBAP (now Bioversity International). p. 45.
  • ReproTech Limited (2012). "Fertility Preservation". ReproTech Limited. Archived from the original on 2012-09-04.
  • Nakasone KK, Peterson SW, Jong SC (2004). "Preservation and distribution of fungal cultures.". Biodiversity of fungi: inventory and monitoring methods. Amsterdam: Elsevier Academic Press. pp. 37–47.
  • Perry SF (1995). "Freeze-drying and cryopreservation of bacteria". Methods in Molecular Biology. Clifton, N.J. 38: 21–30. doi:10.1385/0-89603-296-5:21. PMID 7647859.

External links

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 cryogenic temperatures (temperatures below −100 °C) through the use of vitrification. 21CM was founded in 1993.

Dr. Gregory M. Fahy, who pioneered the use of vitrification in reproductive cryopreservation, serves on the company's Board of Directors and prioritizes, develops and directs the company's research activities. He also manages all extramural collaborative research projects with universities, industry and research institutions to create specific products and services.

The company holds a number of patents, most notably for cryoprotectant mixtures that greatly reduce ice formation while minimizing cryoprotectant toxicity, as well as for synthetic ice-blockers that inexpensively simulate the antifreeze protein found in arctic organisms. Their website lists peer-reviewed journal publications based on research conducted in their laboratories. In 2004 21CM received a $900,000 grant from the U.S. National Institutes of Health (NIH) to study a preservation solution developed by the University of Rochester in New York for extending simple cold storage time of human hearts removed for transplant.At the July 2005 annual conference of the Society for Cryobiology, 21st Century Medicine announced the vitrification of a rabbit kidney to -135 °C with their proprietary vitrification mixture. The kidney was successfully transplanted upon rewarming to a rabbit, the rabbit being euthanized on the 48th day for histological follow-up.On February 9, 2016, 21st Century Medicine won the Small Mammal Brain Preservation Prize. On March 13, 2018, they won the Large Mammal Brain Preservation Prize.

Brian Wowk

Brian G. Wowk, Ph.D. is a medical physicist and cryobiologist known for the discovery and development of synthetic

molecules that mimic the activity of natural antifreeze proteins in cryopreservation applications, sometimes called "ice blockers". As a senior scientist at 21st Century Medicine, Inc., he was a co-developer with Greg Fahy of key technologies enabling cryopreservation of large and complex tissues, including the first successful vitrification and transplantation of a mammalian organ (kidney).

Wowk is also known for early theoretical work on future applications of molecular nanotechnology, especially cryonics, nanomedicine, and optics. In the early 1990s he wrote that nanotechnology would revolutionize optics, making possible virtual reality display systems optically indistinguishable from real scenery as in the fictitious Holodeck of Star Trek. These systems were described by Wowk in the chapter "Phased Array Optics" in the 1996 anthology Nanotechnology: Molecular Speculations on Global Abundance [1], and highlighted in the September 1998 Technology Watch section of Popular Mechanics magazine.

He obtained his undergraduate and graduate degrees from the University of Manitoba in Winnipeg, Canada. His graduate studies included work in online portal imaging for radiotherapy at the Manitoba Cancer Treatment and Research Foundation (now Cancer Care Manitoba), and work on artifact reduction for functional magnetic resonance imaging at the National Research Council of Canada. His work in the latter field is cited by several text books, including

Functional MRI[2] which includes an image he obtained of magnetic field changes inside the human body caused by respiration.

Captive breeding

Captive breeding is the process of maintaining plants or animals in controlled environments, such as wildlife reserves, zoos, botanic gardens, and other conservation facilities. It is sometimes employed to help species that are being threatened by human activities such as habitat loss, fragmentation, over hunting or fishing, pollution, predation, disease, and parasitism. In some cases a captive breeding program can save a species from extinction, but for success, breeders must consider many factors—including genetic, ecological, behavioral, and ethical issues. Most successful attempts involve the cooperation and coordination of many institutions.

Cell bank

A cell bank is a facility that stores cells of specific genome for the purpose of future use in a product or medicinal needs. They often contain expansive amounts of base cell material that can be utilized for various projects. Cell banks can be used to generate detailed characterizations of cell lines and can also help mitigate cross-contamination of a cell line. Utilizing cell banks also reduces the cost of cell culture processes, providing a cost-efficient alternative to keeping cells in culture constantly. Cell banks are commonly used within fields including stem cell research and pharmaceuticals, with cryopreservation being the traditional method of keeping cellular material intact. Cell banks also effectively reduce the frequency of a cell sample diversifying from natural cell divisions over time.

Cryobiology

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

Cryonics

Cryonics (from Greek κρύος kryos meaning 'cold') is the low-temperature freezing (usually at −196 °C or −320.8 °F or 77.1 K) of a human corpse, with the hope that resuscitation may be possible in the future. It is regarded with skepticism within the mainstream scientific community and has been widely characterized as quackery.Cryonics procedures can begin only after clinical death, and cryonics "patients" are legally dead. Cryonics procedures ideally begin within minutes of death, and use cryoprotectants to prevent ice formation during cryopreservation. It is unlikely that a corpse could be reanimated after undergoing vitrification, which causes damage to the brain including its neural networks. The first corpse to be frozen was that of Dr. James Bedford in 1967. As of 2014, about 250 bodies were cryopreserved in the United States, and 1,500 people had made arrangements for cryopreservation after their legal death.

Embryo cryopreservation

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

Ex situ conservation

Ex situ conservation literally means, "off-site conservation". It is the process of protecting an endangered species, variety or breed, of plant or animal outside its natural habitat; for example, by removing part of the population from a threatened habitat and placing it in a new location, which may be a wild area or within the care of humans. The degree to which humans control or modify the natural dynamics of the managed population varies widely, and this may include alteration of living environments, reproductive patterns, access to resources, and protection from predation and mortality. Ex situ management can occur within or outside a species' natural geographic range. Individuals maintained ex situ exist outside an ecological niche. This means that they are not under the same selection pressures as wild populations, and they may undergo artificial selection if maintained ex situ for multiple generations.Agricultural biodiversity is also conserved in ex situ collections. This is primarily in the form of gene banks where samples are stored in order to conserve the genetic resources of major crop plants and their wild relatives.

Fertility preservation

Fertility preservation is the effort to help cancer patients retain their fertility, or ability to procreate. Research into how cancer affects reproductive health and preservation options are growing, sparked in part by the increase in the survival rate of cancer patients.

Greg Fahy

Gregory M. Fahy is a cryobiologist and biogerontologist, and is also Vice President and Chief Scientific Officer at Twenty-First Century Medicine, Inc. Fahy is the world's foremost expert in organ cryopreservation by vitrification. Fahy introduced the modern successful approach to vitrification for cryopreservation in cryobiology and he is widely credited, along with William F. Rall, for introducing vitrification into the field of reproductive biology.In the summer of 2005, where he was a keynote speaker at the annual Society for Cryobiology meeting, Fahy announced that Twenty-First Century Medicine had successfully cryopreserved a rabbit kidney at -130 °C by vitrification and transplanted it into a rabbit after rewarming, with subsequent long-term life support by the vitrified-rewarmed kidney as the sole kidney. This research breakthrough was later published in the peer-reviewed journal Organogenesis.Fahy is also a well-known biogerontologist and is the originator and Editor-in-Chief of The Future of Aging: Pathways to Human Life Extension, a multi-authored book on the future of biogerontology. He currently serves on the editorial boards of Rejuvenation Research and the Open Geriatric Medicine Journal and served for 16 years as a Director of the American Aging Association and for 6 years as the editor of AGE News, the organization's newsletter.

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 for social reasons such as employment or studying. Several studies have proven that most infertility problems are due to germ cell deterioration related to ageing. Surprisingly, the uterus remains completely functional in most elderly women. This implies that the factor which needs to be preserved is the woman's eggs. The eggs are extracted, frozen and stored. The intention of the procedure is that the woman may choose to have the eggs thawed, fertilized, and transferred to the uterus as embryos to facilitate a pregnancy in the future. The procedure's success rate (the chances of a live birth using frozen eggs) varies depending on the age of the woman, and ranges from 14.8 percent (if the eggs were extracted when the woman was 40) to 31.5 percent (if the eggs were extracted when the woman was 25).

Orto Botanico di Cascina Rosa

The Orto Botanico di Cascina Rosa (about 22,000 m²) is a botanical garden maintained by the University of Milan, and located at the end of Via Carlo Valvassori Peroni, Milan, Italy. It is open daily.

The garden was established on disused farmland in 2002 for research and education. Its primary research facilities are three greenhouses that include a total of 10 separate compartments that support modern technology including cryopreservation, molecular testing, etc. Current research includes genetic improvement of rice, and exploration of useful genes in Arabidopsis thaliana. The garden's grounds contain many labeled plants, lawns, a lake, and about 1 km of walking paths.

Ovarian tissue cryopreservation

Ovarian tissue cryopreservation is cryopreservation of tissue of the ovary of a female.

Ovary

The ovary is an organ found 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 (from Latin ovarium, meaning 'egg, nut') found on the left and right sides of the body. The ovaries also secrete hormones that play a role in the menstrual cycle and fertility. The ovary progresses through many stages beginning in the prenatal period through menopause. It is also an endocrine gland because of the various hormones that it secretes.

Raffinose

Raffinose is a trisaccharide composed of galactose, glucose, and fructose. It can be found in beans, cabbage, brussels sprouts, broccoli, asparagus, other vegetables, and whole grains. Raffinose can be hydrolyzed to D-galactose and sucrose by the enzyme α-galactosidase (α-GAL), an enzyme not found in the human digestive tract. α-GAL also hydrolyzes other α-galactosides such as stachyose, verbascose, and galactinol, if present. The enzyme does not cleave β-linked galactose, as in lactose.

The raffinose family of oligosaccharides (RFOs) are alpha-galactosyl derivatives of sucrose, and the most common are the trisaccharide raffinose, the tetrasaccharide stachyose, and the pentasaccharide verbascose. RFOs are almost ubiquitous in the plant kingdom, being found in a large variety of seeds from many different families, and they rank second only to sucrose in abundance as soluble carbohydrates.

Humans and other monogastric animals (pigs and poultry) do not possess the α-GAL enzyme to break down RFOs and these oligosaccharides pass undigested through the stomach and upper intestine. In the lower intestine, they are fermented by gas-producing bacteria that do possess the α-GAL enzyme and make carbon dioxide, methane or hydrogen—leading to the flatulence commonly associated with eating beans and other vegetables. α-GAL is present in digestive aids such as the product Beano.

Research has shown that the differential ability to utilize raffinose by strains of the bacteria Streptococcocus pneumoniae, impacts their ability to cause disease.

Procedures concerning cryopreservation have used raffinose to provide hypertonicity for cell desiccation prior to freezing. Either raffinose or sucrose is used as a base substance for sucralose.

Saul Kent

Saul Kent is a life extension activist, and co-founder of the Life Extension Foundation, a dietary supplement vendor and promoter of anti-aging research. He is also a pioneer in the practice of cryonics, and was a board member of the cryonics organization Alcor Life Extension Foundation.

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. For human sperm, the longest reported successful storage is 24 years. 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.

Thomas K. Donaldson

Thomas K. Donaldson (1944–2006) was a mathematician and well-known cryonics advocate. He was born in the state of Kentucky in the United States, and took his Ph.D. from the University of Chicago in 1969. He also lived in Sunnyvale, California, and for many years in Canberra, Australia, where he taught mathematics at Australian National University. He founded both the Cryonics Association of Australia and the Institute for Neural Cryobiology, which has funded ground-breaking research in cryopreservation of brain tissue.

In 1976 Donaldson published A Brief Scientific Introduction to Cryonics [1], the first concise review of scientific literature supporting the practice of cryonics. He was a regular contributor to Cryonics magazine, the newsletter of the Alcor Life Extension Foundation, for many years. He also published his own periodical, Periastron, which discussed neuroscience issues as they pertain to cryonics.

Donaldson proposed some of the earliest ideas for cell repair technologies, seeing such technologies as extensions of natural biology, but using new enzymes and solvents other than water for low temperature operation. When Eric Drexler’s ideas about molecular nanotechnology came to dominate cryonics thinking in the mid-1980s, he frequently expressed concern that too much reliance was being placed on the new molecular-mechanical repair paradigm to the exclusion of earlier biological approaches. Donaldson’s seminal exposition of his vision of future medicine was his 1988 essay, 24th Century Medicine [2].

The views expressed by Donaldson on the subject of death were far reaching even by cryonics standards [3]. According to Donaldson, as long as the brain continues to exist in some kind of repairable form, “death” was merely a label indicating that the memory and personality information within it were beyond reach of current technology. While all cryonics proponents would agree with that where today’s technology is concerned, Donaldson went further. Instead of expecting a plateau of “mature nanotechnology” to someday clearly answer whether cryopreserved patients are information theoretically dead, he suggested that increasingly sophisticated methods for decrypting the original information content of injured brains would always keep coming. He wrote of “neural archaeology” as an important part of future medicine. He said cryonics in some form would always be necessary because whether certain brain injuries were ultimately repairable would always remain an open question for the future.

Donaldson also maintained an avid interest in biomedical gerontology, self-publishing the book "A Guide to Anti-aging Drugs" in 1994. Despite this interest, he was pessimistic about near-term prospects for extension of human lifespan. In 1986 he stated that only small children might live long enough to see advances allowing them to avoid the need for cryonics. In late 2005, he wrote in Cryonics magazine, "We aim, by cryopreservation, to reach a time when aging can be reversed and abolished. Cryopreservation may well turn out to be the only way that anyone (now living) has any chance of doing that."

In 1988, Donaldson was diagnosed with grade II astrocytoma, a type of malignant brain tumor. Despite radiation therapy, his long-term prognosis was poor. In 1990 he received international attention when he unsuccessfully sued the Attorney General of the State of California for the right to an elective cryopreservation to prevent the tumor from destroying his brain [4]. An episode of the television drama L.A. Law was based on his story. Although he was criticized for wanting to sacrifice life today for uncertain life in the future, the intent of his lawsuit was to obtain the right to cryopreservation should his tumor begin regrowing, not a desire for immediate cryopreservation.

In early 2006, his friend Steve Bridge posted a message to the Cryonet email list indicating that Donaldson’s cancer had returned, and that he was returning from Australia to the United States in serious condition. He is

cryopreserved at the Alcor Life Extension Foundation; his biography matches the description of patient A-1097, described in the Spring 2006 issue Cryonics Magazine, who received an unusually smooth cryopreservation on January 19, 2006.

Vitrification

Vitrification (from Latin vitreum, "glass" via French vitrifier) is the transformation of a substance into a glass, that is to say a non-crystalline amorphous solid. In the production of ceramics, vitrification is responsible for its impermeability to water.Vitrification is usually achieved by heating materials until they liquidize, then cooling the liquid, often rapidly, so that it passes through the glass transition to form a vitrified solid. Certain chemical reactions also result in glasses.

In terms of chemistry, vitrification is characteristic for amorphous materials or disordered systems and occurs when bonding between elementary particles (atoms, molecules, forming blocks) becomes higher than a certain threshold value. Thermal fluctuations break the bonds; therefore, the lower the temperature, the higher the degree of connectivity. Because of that, amorphous materials have a characteristic threshold temperature termed glass transition temperature (Tg): below Tg amorphous materials are glassy whereas above Tg they are molten.

The most common applications are in the making of pottery, glass, and some types of food, but there are many others, such as the vitrification of an antifreeze-like liquid in cryopreservation.

In a different sense of the word, the embedding of material inside a glassy matrix is also called vitrification. An important application is the vitrification of radioactive waste to obtain a substance that is hopefully safer and more stable for disposal.

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