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As we known penetrating cryoprotectants acts as colligatively meaning if we added cryoprotectants such as DMSO (dimetyl solfoxide), the freezing point of liquid ( a solvent) is depressed which can caused a solution has a lower freezing point than a pure solvent. In other words, it can lowering the temperature of the solution which is good for cryopreservation work because cells need to be frozen until it reach glass transition temperature where cells already vitrified to allow solidification without any crystal ice formation inside the cells. Properties for each cryoprotectants and why they are choosed Normally during cryopreservation, penetrating colligative cryoprotectants such as DMSO, glycerol, polyethylene glycol and ethylene glycol are mostly being used to reduce cell damage. These cryoprotectents are choose to be used can be referring by Tao and Li (1986) which classifying a range of frequently used plant cryoprotectants on their ability to penetrate the cells. They are cryoprotectants that can penetrate through the cell wall only (oligosaccharides, mannitol amino acids (proline) and low molecular weight polymers such as PEG or polyethylene glycol) and another one is cryoprotectants that can penetrate through the cell wall and membrane such as DMSO and glycerol. DMSO has proved to be an excellent cryoprotectant because of its goods properties too. It has a low molecular weight, is easily miscible with the solvent, non toxic at low concentration, easily permeable into the cells and easily washable from the cells which all this properties to prevent plasmolysis and protect cells against osmotic shocks. A better concentration for DMSO generally at 5-10 % and 10-20% for glycerol is adequate for most materials. Application of single cryoprotectants doesn’t result in high survival a mixture of cryoprotectants has proven beneficial. For example normally in cryopreservations of plant cells, PVS2 will be used as additives. The purposes of PVS2 cryoprotectant mixture which penetrating cryoprotective such as DMSO is one of the mixtures are to replaces cellular water inside cell and it can changes the freezing behavior of water remaining in the cells. Besides, function of penetrating cryoprotectants in PVS2 mixture are also to replace water as the cells become dehydrated and prevent injurious cell shrinkage caused by dehydration itself (Volk and Walters 2006). How cryoprotectants works If we added penetrating cryoprotectants in cell solution, it will may cause temporary plasmolysis in plant cell where water flow out from the cell because cell solution become increasingly concentrated extracellular and less concentrated inside the cell. Cryoprotectants will penetrate the wall and loosen adhesion between cell wall and cell membrane. Since penetrating cryoprotectants enter the cell, cell become concentrated intracellularly because addition of cryoprotectant also increase the osmolality inside the cell which cryoprotectant replace the water molecules and can cause the reducing of moisture content inside the cell due to dehydration of cell occurred. This situation can cause plant cells vitrified (vitrification process happens) because some unfrozen liquid is too viscous and highly concentrated to permit ice crystal growth. It shows that plant cell achieve glass transformation temperature usually below -40 which ice turn into solid glassy state by homogenous nucleation during slow cooling. Thus, we can avoid intercellular ice formation too because since penetrating cryoprotectants entering the cells, it can reduce cell damage. Some of the cryoprotectants such as polyethylene glycol that only penetrate the wall not the plasma membrane can prevent ice crystals from damaging the cell membrane and also buffer the cytoplasm from excessive dehydration. While some cryoprotectant such as non penetrating cryoprotectants that remain in intercellular spaces and do not penetrate either structure become concenterated at the ice crystal front, it will inhibit the rate and extent of ice growth and protect from mechanical deformation. Toxicity of cryoprotectants As we know, good and effective cryoprotectants must be non toxic to the cell at high concentration, so that the cryoprotectants must be added with combination of others components. Fahy et al. (1986) show that it is more better and effective to use a mixture of additives in order to lower the toxicity of any single additives, limits the impacts of extreme evaporative drying and also helps to stabilize the glassed formed. He also recommended using mixture of penetrating and non penetrating cryoprotectants. Frinkle and Ulrich (1979) suggested that using more than one kind of cryoprotectant can dilute concentration of one cryoprotectant and then reduce the toxicity of cryoprotectants to the cells. By referring to the journal of effects of cryoprotectants on cryopreservation of sugarcane done in 1993, the presence of DMSO at 10% concentration showed no growth of sugarcane and when treated by 15% DMSO showed poor growth after plunging into liquid nitrogen. The best combination of cryoprotectants used was 15% DMSO with 1.0 sucrose which showed moderate poor growth after plunging into liquid nitrogen. For example, a single cryoprotectants such as dimetyl sulfoxide (DMSO) is effective but the combination of dimetyl sulfoxide (DMSO) with glycerol (each at 0.5M) and a third component such as sucrose, proline,mannitol or sorbitol (at 1.0M)give more effective than single cryoprotectant. The cryoprotectants mixture has been used for vitrification where solidification occur without any ice crystal formation and intracellular ice formation (IIF).