**3. Cryoprotectants**

CPAs play key roles in protecting the vitality of the cryopreserved cells during their processing and storage at ultralow temperature and their subsequent recovery with normal physiological functionality. Christopher Polge and his colleagues discovered the cryoprotective capabilities of glycerol in the late 1940s that subsequently led to the successful cryopreservation of cattle and poultry spermatozoa [17]. This discovery introduced a fascinating branch of bio-physical science, the cryobiology. CPAs are water-soluble chemical substances with low level of cytotoxicity that lower the melting point of water. CPAs can be divided into two categories, membrane permeating and membrane non-permeating.

 Membrane-permeating CPAs are small molecules that easily penetrate the cell membranes. During cryopreservation, these agents decrease freezing point and prevent cell damage from high electrolyte concentrations. They form linkages with the electrolyte molecules and thus act as partial substitute to water [18]. Penetrating CPAs also stabilize lipid membranes by hydrogen bonding with the membrane lipids, which is especially important under severely dehydrated conditions.

On the other hand, the non-permeating CPAs increase the viscosity of the cryopreservation solution generating osmotic gradient across the cell membrane and thus withdraw intracellular water [19]. These agents allow effective dehydration of the cells even in the presence of permeating CPAs at low concentration. Additionally, non-permeating CPAs also reduce mechanical stress that occurs during cryopreservation [20]. The list of CPAs commonly used for cryopreservation of oocytes and embryos is provided in **Table 2**.

 It may be noted that no permeating CPA is completely devoid of the capability to induce cell toxicity. Therefore, the use of a single permeating CPA substantially increases the possibility of cellular toxicity to the frozen cells, because of their high concentration in the cryopreservation solution. In contrast, similar viscosity of cryopreservation solution can be achieved by using a combination of permeating CPAs along with non-permeating cryoprotective agents. In the latter case, the


**Table 2.** 

*Commonly used cryoprotectants for cryopreservation of oocytes and embryos.* 

concentration of a particular permeating CPA would be much lower in the solution. Therefore, it is believed that the CPA-mediated toxicity to the cells can be reduced if a combination of CPAs is used for cryopreservation.
