Vitrification, Preservation and Cryoprotectants

We all know that living organisms are primarily composed of water and on freezing, these water molecules convert into ice. There are many amphibians and insects who can withstand freezing without converting into ice. Amphibians like frog produce glycerol by the liver and it works like ‘antifreeze’. Glycerol, ethylene glycol etc. are cryoprotectants and prevent ice formation. The cryoprotectant prevents water from forming into crystals and makes it harden like glass. This is known as Vitrification. At freezing temperature, normal cells get damaged due to ice crystal formation. When a cryoprotectant is used the water molecules get frozen in their own locations without forming the ice crystal lattice. This thus causes no damage to the cell and thus the cells, tissues and even whole organs can be maintained in that particular biological stage without causing any damage to it.

Cryoprotectants work by reducing ice formation, but there are substances which are known by the name of ice blockers which are targeted towards ice nuclei formation and thus stop freezing. This was first observed in artic fish which used certain anti-freeze proteins to prevent freezing. To bring about Vitrification, cryoprotectantshave to be used in very high concentrations which might at times lead to toxicity. The problem was that cryoprotectants became too viscous at high concentrations making perfusion difficult. This problem was effectively solved by using ice-blocker along with cryoprotectant to bring about optimal vitrification.

Glycerol was the first ever cryoprotectant used initially to freeze store bull sperm and was later adopted to store red blood cells. Later DiMethyl Sulfoxide was used for vitrification where it was used along with glycerol and slow cooling technique to store mouse embryos which were later rewarmed to produce live mice. The breakthrough in vitrification storage was when the 8 cell human embryo was preserved at liquid nitrogen temperature by using the optimal concentrations of DMSO at the several stages. Human embryos are now preserved at 4 and even 2 celled stages using a combination of DMSO, glycerol and propylene glycol. Today, over a million embryos are being preserved worldwide and have been proven to be a giant leap towards infertility treatment. There are several children round the world who have been once cryoprotected embryos at liquid nitrogen temperature.

Glycerol was used effectively for cryopreservation of human blood and sperm and was also used to prevent freeing in cryonics patients. The problem was due to the viscosity of glycerol in high concentrations that it could not bring about complete vitrification. The problem was well realized when the need came up to vitrify tissues and organs. Glycerol as cryoprotectant could only bring about partial vitrification with 20% ice formation. Some organs can become completely damaged even by small ice crystals as it affects the connection between the cells. Crystallization when using glycerol for vitrification can be prevented by using glycerol solution of the optimal concentration of 68%volume/volume glycerol and water solution. At this concentration there was no crystallization that occurred and water hardened like glass.

The devitrification problem is caused mainly due to the rewarming of a cryoprotected tissue which leads to ice crystal formation. This can cause damage to the tissues, organs or cells on re-obtaining the cells from its vitrified stage. This devitrification problem can be prevented by rewarming at high temperatures quickly. According to reports, researches and calculations, to bring about rewarming without devitrification problem, it is required to deliver 1667 degree temperature in a sec. This high and quick temperature to retrieve vitrified cells can be obtained through radio frequency rewarming. Anti-nucleators like polyvinyl alcohol can also bring about rewarming without causing destruction to the cells.