Cryopreservation

Cryopreservation is the extended storage of tissues, cells or nuclei at low temperatures. Cryopreservation allows storage for an indefinite time (greater than 50,000 years) in liquid nitrogen at a temperature of -196 degrees Centergrade. The survival of cells during cryopreservation and thawing depends on the cooling and warming rates.

The left hand side of the diagram shows as sperm cells are cooled from room temperature a number of harmful processes can occur. Some cells suffer by being cooled (cooling injury), these cells benefit from rapid cooling to just above freezing temperature. As unprotected cells reach freezing temperatures damage can occur as they dehydrate (loose water) to the surrounding ice. If cooling is uncontrolled large ice crystals can form in the cells or tissue and these can also cause damage.

To minimise the potential amount of ice that may form during freezing, cells are PERMEATED with CRYOPROTECTANTS (right hand side of diagram) before freezing. Cryoprotectants are compounds which replace water within cells without causing too greater toxicity. They allow dehydration of the cell without to much shrinkage and preserve cell cytoplasm and organelles. Common cryoprotectants are glycerol and dimethyl sulphoxide.

 

Under CONTROLLED COOLING the cells are frozen at controlled rates which are optimised for cell survival by controlling the size and amount of ice crystals. These ice crystals grow between the cytoplasm of the cell and do not cause lethal damage. For example in the cryopreservation of frog sperm a series of cooling rates such as 0.3 degrees Centergrade per minute from -8 to -16 degrees, 3.0 degrees from -8 to -16, and 5 degrees from -16 to -80 degrees are used to control ice crytal growth (see diagram). At -80 degrees the sperm cells are then quenched in liquid nitrogen for permanent storage.

Under VITRIFICATION, rapid cooling by quenching in liquid nitrogen to form a glass like structure, even with cryoprotectants a large number of very small ice crystals form within the cells. During thawing these small ice crystals grow throughout the cells and cause damage. However, this technique works with some cells if the thawing rates are high.

Size and Complexity

In 1946 sperm was first cryopreserved by Rostand using egg yolk and glycerol as a cryoprotectant. This breakthrough was serindipidous as the cryoprotectant was a common mixture used to fix tissues to slides and was accidentally mixed with the sperm. The researchers only identified the "mistake" when the mixture fell on a hot plate ane they recognised the smell. Since then the size and complexity of the cells and tissues that can be cryopreserved has increased. Now the larvae of many aquatic organisms and insect larvae, and some small organs, can be cryopreserved. The maximum size of these is about 1mm.

However, insect larvae such as mosquito larvae and the larvae of vertebrates (tadpoles and fish) have proved impossible to revive after freezing. The main reasons are the difficulty of achieving adequate cryoprotectant permeation without lethal effects, and unequal freezing and thawing.