Cryopreservation.html

Cryopreservation of plant shoots. Open tank of Liquid Nitrogen behind.

A tank of liquid nitrogen, used to supply a cryogenic freezer (for storing laboratory samples at a temperature of about −150 degrees Celsius).

Cryopreservation is a process where cells or whole tissues are preserved by cooling to low sub-zero temperatures, such as (typically) 77 K or −196 °C (the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped. However, when vitrification solutions are not used, the cells being preserved are often damaged due to freezing during the approach to low temperatures or warming to room temperature.

1 Risks
2 Prevention of risks
3 Freezable tissues
4 Natural cryopreservation
5 History
6 See also
7 References
8 External links

Risks

Phenomena which can cause damage to cells during cryopreservation are solution effects, extracellular ice formation, dehydration and intracellular ice formation. Many of these effects can be reduced by cryoprotectants.

Solution effects

Solution effects caused by concentration of solutes in non-frozen solution during freezing as solutes are excluded from the crystal structure of the ice. High concentrations 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

The 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.

Prevention of risks

Controlled-Rate and Slow Freezing in Cryopreservation is a well established technique pioneered in the early 1970s which enabled the first human embryo frozen birth in 1984 (Zoe Leyland http://www3.interscience.wiley.com/journal/118746246/abstract). Since then machines that freeze biological samples using programmable steps, 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 deep frozen, or cryopreserved, in liquid nitrogen. Such machines are used for freezing oocyte, skin, blood products, embryo, sperm, stem cells and general tissue preservation in hospitals, veterinary practices and research labs around the world. As an example, estimates put the number of live births from frozen embryos 'slow frozen' at some 300,000 to 400,000 or 20% of the estimated 3 million IVF births. (http://www.bionews.org.uk/commentary.lasso?storyid=4055 )

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 were presented at the American Society for Reproductive Medicine conference in San Francisco, US, 2008. The studies indicate that using frozen embryos rather than fresh embryos reduced the risk of stillbirth and premature delivery though the exact reasons are still being explored. http://www.asrm.org/Professionals/Meetings/sanfrancisco2008/Abstracts2008.pdf

Vitrification a new technique, claims to provide the benefits of cryopreservation without damage due to ice crystal formation. In clinical cryropreservation, vitrification usually requires the addition of cryoprotectants prior to cooling. The cryoprotectants act like antifreeze: they lower the freezing temperature. They also increase the viscosity. Instead of crystallizing, the syrupy solution turns into an amorphous ice — i.e. it vitrifies. Vitrification of water is promoted by rapid cooling, and can be achieved without cryoprotectants by an extremely rapid drop in temperature (megakelvins per second). The rate that is required to attain glassy state in pure water was considered to be impossible until recently.¹

Two conditions usually required to allow vitrification are an increase in the viscosity and a depression of the freezing temperature. Many solutes do both, but larger molecules generally have larger effect, particularly on viscosity. Rapid cooling also promotes vitrification.

In artificial cryopreservation, the solute must penetrate the cell membrane in order to achieve increased viscosity and depressed 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 high concentration. One of the difficult compromises faced in artificial cryopreservation is limiting the damage produced by the cryoprotectant itself.

Freezable tissues

In general, cryopreservation is easier for thin samples and small clumps of individual cells, because these can be cooled more quickly and so require lower doses of toxic cryoprotectants. Therefore, the goal of cryopreserving human livers and hearts for storage and transplant is still some distance away.

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

Semen (which can be used successfully almost indefinitely after cryopreservation),
Blood (special cells for transfusion, or stem cells)
Tissue samples like tumors and histological cross sections
Human eggs (oocytes) See oocyte cryopreservation
Human embryos that are 2, 4 or 8 cells when frozen (pregnancies have been reported from embryos stored for 9 years. Many studies have evaluated the children born from frozen embryos, or “frosties”. The result has uniformly been positive with no increase in birth defects or development abnormalities.)[1]

In addition, efforts are underway to preserve humans cryogenically, known as cryonics. In such efforts either the brain within the head or the entire body may undergo the above process. Cryonics is in a different category from the aforementioned examples, however, for while countless cryopreserved cells, vaccines, tissue and other biologial samples have been thawed and successfully used, 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 make a case 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 vastly advanced future technology.

Natural cryopreservation

Water bears (or tardigrada), microscopic multicellular organisms, can survive freezing at low temperatures by replacing most of their internal water with the sugar trehalose. Sugars and other solutes that do not easily crystallize have the effect of limiting the stresses that damage cell membranes. Trehalose is a sugar that does not readily crystallize. Mixtures of solutes can achieve similar effects. Some solutes, including salts, have the disadvantage that they may be toxic at high concentrations.

History

One of the most important early workers on the theory of cryopreservation was James Lovelock of Gaia theory fame. Dr. Lovelock's work suggested that damage to red blood cells during freezing was due to osmotic stresses. Lovelock in early 1950s had also suggested that increasing salt concentrations in a cell as it dehydrates to lose water to the external ice might cause damages to the cell.². Cryopreservation of tissue in recent times started with the freezing of fowl sperm, which in 1949 was cryopreserved for the first time by a team of scientists in the UK led by Dr Christopher Polge. The process moved into the human world in the 1950s with pregnancies obtained after insemination of frozen sperm. However the rapid immersion of the samples in liquid nitrogen did not, for certain of these samples–such as types of embryos, bone marrow and stem cells–produce the necessary viability to make them usable on thawing. Increased understanding of the mechanism of freezing injury to cells emphasised 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 concentration of solutes as progressively more ice is formed. In 1963 Peter Mazur, at Oak Ridge National Laboratory in the USA, showed 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.

References

^ Bhat SN, Sharma A, Bhat SV (2005). "Vitrification and glass transition of water: insights from spin probe ESR". Phys Rev Lett 95 (23): 235702. doi:10.1103/PhysRevLett.95.235702. PMID 16384318.
^ Mazur P (1970). "Cryobiology: the freezing of biological systems". Science 168 (934): 939–49. doi:10.1126/science.168.3934.939. PMID 5462399.

3. Engelmann, F., M.E. Dulloo, C. Astorga, S. Dussert and F. Anthony, editors (2007). Conserving coffee genetic resources. Bioversity International, CATIE, IRD. http://www.bioversityinternational.org/Publications/pubfile.asp?ID_PUB=1244. 61 p.

4. Panis, B and Tien Thinh, N. (2001). Cryopreservation of Musa germplasm. INIBAP (now Bioversity International). http://www.bioversityinternational.org/Publications/pubfile.asp?ID_PUB=722. 45 p.