Cryo-preservation

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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 (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. 

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. 

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.

FREEZABLE TISSUES

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, 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:

  • 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.
    • Umbilical cord blood 
  • Tissue samples like tumors and histological cross sections
  • Eggs (oocytes) in oocyte cryopreservation
  • Embryos at cleavage stage (that are 2, 4 or 8 cells) or at blastocyst stage, in embryo cryopreservation
  • Ovarian tissue in ovarian tissue cryopreservation
  • Plant seeds or shoots may be cryopreserved for conservation purposes.