Vitrification versus conventional cryopreservation technique

During in vitro fertilization treatment (IVF), a surplus of oocytes is produced which results in a surplus of embryos which must be preserved. Due to the recent tendency in ART treatment to transfer fewer embryos in order to avoid multiple pregnancies, partly a result of new laws and guidelines aimed at controlling the number of embryos to be transferred and the number of oocytes to be fertilized, the need for simpler, more effective methods for the cryopreservation of embryos and oocytes is increasing. Currently, zygotes and embryos are typically cryopreserved by means of the traditional slow-rate freezing protocol, while oocyte freezing is still in the experimental phase. However, during the slow freezing method, ice crystals are formed or produced, which has a deleterious effect on the cells. Intracellular ice crystals can damage the cell wall and structure, while the extracellular precipitation of water as ice crystals increases the salt concentration to levels that cause damage to the cell. A delicate balance between these potentially harmful factors must be maintained throughout the slow freezing process to ensure survival of the cells. In 1985, the ultra-fast freezing method through vitrification was introduced based on inducing solidification of the cells and the surrounding vitrification solution (glass-like or vitreous) without formation of any ice crystallization. This can be achieved through the combination of the following factors: 1Increasing the concentration of cryoprotectant: The cryoprotectant is usually used in much higher concentration than in the slow freezing method. To avoid the toxic effect of the cryoprotectant, cells are equilibrated in a low concentration solution before immersion in the high concentration solution (vitrification solution). The equilibration solution contains between 10 to 15% of the cryoprotectant while the vitrification solution contains between 35 and 40% (around 5.5 Molar) of the cryoprotectant. Selecting the optimal cryoprotectant is critical. It should be of high permeability (low molecular weight) and as nontoxic to the cells as possible. Ethylene Glycol (EG) has these characteristics and, to lesser extent, also DMSO. 2Dehydration of the cells: This is achieved by using a high concentration of nonpermeating cryoprotectant of a large molecular weight such as disaccharide sugars: sucrose or galactose at a concentration up to 1 Molar. The end concentration of the nonpermeating cryoprotectant is added to the vitrification solution. We cannot overemphasize the importance of minimizing the cells' exposure time in the highly concentrated vitrification solution; successful vitrification procedures must limit such exposure to a few seconds. 3Very fast freezing rate: The vitrification device containing the cells is directly plunged into liquid nitrogen which can achieve a cooling rate of more than 20,000°C/ minute. The rate depends on the vitrification device used and on the freezing volume. No special equipment is needed to achieve such a freezing rate. When the vitrification device enters the liquid nitrogen, a