Cryopreservation

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.

Risks

Phenomena which can cause damage to cells during cryopreservation are solution effects, extracellular ice formation, dehydration and intracellular ice formation.

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 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

Vitrification provides the benefits of cryopreservation without the 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.

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 rapid 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 many cryopreserved cell suspensions, thin tissue samples, and some small organs have been warmed and successfully used, this has not yet been the case for cryopreserved brains or bodies. 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.

References

3. Engelmann, F., M.E. Dulloo, C. Astorga, S. Dussert and F. Anthony, editors (2007). Conserving coffee genetic resources. Bioversity International, CATIE, IRD.  61 p.

4. Panis, B and Tien Thinh, N. (2001). Cryopreservation of Musa germplasm. INIBAP (now Bioversity International).  45 p.