Cryopreservation: A Technique of Freezing

Cryopreservation is a technique of freezing tissues or cells to preserve for use at a later date. Cells are usually stored at ultra-low temperatures below – 1960C or 77K (boiling point of liquid nitrogen because at this low temperature, any biological activities, including the biochemical reactions that would lead to cell death, is effectively stopped. Hence the tissues can be stored for several years. This preservation can be made by the use of cryoprotectants.


The cryoprotectants are generally used to protect the biological tissues from freezing and damage from ice formation. They act like antifreeze by lowering the freezing temperature and increasing the viscosity of the cell. They are mainly divided into two broad classes

Penetrating cryoprotectants and non-penetrating cryoprotectants. Mechanism(s) of protection by cryoprotectants – I

Penetrating agents

The mechanism of cryoprotectants was 1st proposed by Lovelock. He explained that the mechanism of action of cryoprotectants was due to colligative properties. The colligative property of a solution does not depend on the number of the solvents but it depends on the concentration of the molecules. For e.g.; dmso cream uses (Dimethyl sulfoxide).There are also other penetrating cryoprotectants which are commonly used, such as propylene glycol, ethylene glycol, glycerol.

Mechanism(s) of protection by cryoprotectants – I

Non-penetrating agents

These cryoprotectants are thought to act by dehydrating the cell at high sub-freezing temperatures. These compounds are generally polymers that form extensive hydrogen bonds with water, reducing the water activity, for e.g.- Hydroxy-ethyl-starch and other non-penetrating cryoprotectants are used such as Polyvinyl Pyrrolidone (PVP),

Polyethylene Oxide (PEO).

Cooling rate

Most cultured cells survive best if they are cooled at 10C/min, but if recovery is low, we should change the freezing rate (that is we should use more or less insulation).


There are there main types of liquid-nitrogen system. They are- narrow-necked with ampules on canes in canisters (high capacity, low temperature), wide-necked with ampules in triangular racks ( high capacity, high boil-off), narrow-necked with ampules in square racks (moderate capacity, low boil-off rate).

Storage of the tissue or the cell

The storage of the tissues are done in two phases, in vapor phase and in liquid phase. The

storage in the vapors phase eliminates the risk of explosion with sealed ampules while the storage in the liquid phase the container can be filled with the liquid nitrogen and therefore the cells last longer.

What is preserved?

The tissues that are to be preserved are mainly biological materials, particularly cell suspensions or thin tissue samples. For e.g. semen (which can be used successfully after cryopreservation, blood (special cells for transfusion, or stem cells),tissue samples like tumors and histological cross sections, eggs (Oocytes). Recovery Thawing

Usually rapid thawing should be done to avoid damage from ice crystal growth and then the tissues should be immediately immersed in water bath and should be transferred to re-warmed media. Excess alkalinity must avoided, dish should be placed in the incubator to allow the saturation with CO2. The thawed cells must be washed of cryoprotectants within 24 hours and nursed back to normal growth. Now the materials should be diluted slowly.

Advantages of cryopreservation

The cryopreservation technique can maintain the preserved materials without any constant care and hence it saves the time as well as the reagents. It reduces the risk of microbial contamination and here the cross contamination with other cell lines is very rare. It can reduce the rate of genetic drift and morphology. The freezing embryos are very much advised for the women who are at a high risk of developing severe ovarian hyper stimulation syndrome and frozen embryos have also become available for donation to infertile couples. The stored tissues are very much useful for research purposes and it has proved to be boon for endangered species.

Role of cryopreservation in biodiversity conservation

In the present scenario wild life is facing the threat of extinction due to habitat destruction. Therefore, conservation of habitat as well as germplasm is the present day challenge for the scientists. The concept of Frozen Zoo since 1980s proved to be a boon for ex-situ conservation. The germplasm of endangered species as well as the plants and microbes can be stored by cryopreservation for future production in captive conditions and they can be conserved. To site a few example; in 1972, the first mammal (mouse) was born from frozen embryo and in 1984, the first human baby was born from frozen embryo transfer.

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