SEMEN CRYOPRESERVATION

Cryopreservation or cryoconservation is a process where cells, whole tissues, or any other substances susceptible to damage caused by chemical reactivity or time are preserved by cooling to sub-zero temperatures.

In embryology lab there are two techniques that used for freezing of sperm , egg, embryos and ovarian tissues . Slow Freezing and Vitrification

PRINCIPLES OF CRYOBIOLOGY

A basic principle of cryobiology is that the extent of freezing damage depends of the amount of the free water in the system and the ability of that water to crystallize during freezing. During freezing, most of the water changes to ice, and cellular metabolism ceases. Cryopreservation can be done using conventional slow freezing techniques or by newer techniques as vitrification.

assited-embryo-hatching

Conventional slow cooling:

It is based on the principle of dehydration, where cooling rates are optimized to remove water from the embryo, preventing cryoinjury from ice crystal formation while minimizing chemical toxicity, and osmotic stress from exposure to high concentration of salts.

Two common cryoprotective agents are dimethyl sulfoxide (DMSO) and glycerol. A sugar called trehalose, which occurs in organisms capable of surviving extreme dehydration, is used for freeze-drying methods of cryopreservation. Trehalose stabilizes cell membranes, and it is particularly useful for the preservation of sperm, stem cells, and blood cells.

Most systems of cellular cryopreservation use a controlled-rate freezer. This freezing system delivers liquid nitrogen into a closed chamber into which the material to be frozen is placed. Careful monitoring of the rate of freezing helps to prevent rapid cellular dehydration and ice-crystal formation. In general, the cells are taken from room temperature to approximately −90 °C (−130 °F) in a controlled-rate freezer. The frozen cell is then transferred into a liquid-nitrogen freezer maintained at extremely cold temperatures with nitrogen in the liquid phase.
Vitrification :

It is now regarded as a potential alternative to the conventional slow freezing method. It is a process of rapid cooling of a liquid by short exposure with a high concentration of CPA to achieve a glass like solid state. It has the advantage of preventing ice crystal formation and this rapid freezing technique minimizes chilling injury and osmotic shock to the embryo. Two conditions usually required to allow vitrification are an increase in the viscosity and a depression of the freezing temperature. These conditions are achieved by addition of cryo protective agents.[CPA] which acts like antifreeze. Vitrification usually requires the addition of CPA prior to cooling. Other components required for vitrification are basal medium buffered with phosphate or HEPES, macromolecules, sugars and proteins. Vitrification was first used for preserving embryos from the mouse in 1985. Since then the techniques has undergone various improvements by using less toxic and more permeable chemicals, by using combination of CPA to reduce toxicity, using a stepwise approach to equilibration and increasing cooling and warming rates.
Ethylene glycol has been widely used as a CPA in both slow freezing and vitrification methods due to its high permeation ability and low toxicity compared with other CPAs.

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