蔗糖在冷凍劑中扮演提高滲透壓, 將細胞內水分脫出, 縮短抗凍劑EG, DMSO滲透入細胞內之時間, 減少抗凍劑EG, DMSO毒性

蔗糖在解凍劑中扮演滲透壓緩衝劑, 藉維持細胞外高滲透壓, 避免水分快速滲入細胞內, 減少osmotic shock


http://www.intechopen.com/books/recent-advances-in-cryopreservation/the-maining-of-cryopreservation-for-in-vitro-fertilization-patients


Cryoprotectant agents are essential for the cryopreservation of cells. Basically two groups of cryoprotectants exist: 1) permeating (glycerol, ethylene glycol, dimethyl sulphoxide); and 2) nonpermeating (saccharides, protein, polymers) agents. The essential component of a vitrification solution is the permeating agent. These compounds are hydrophilic non-electrolytes with a strong dehydrating effect. Furthermore, these CPAs are able to depress the “freezing point” of the solution. Regarding the high concentration of cryoprotectant used for vitrification, and in view of the known biological and physiochemical effects of cryoprotectants, it is suggested that the toxicity of these agents is a key limiting factor in cryobiology. Not only does this toxicity prevent the use of fully protective levels of these additives, but it may also be manifested in the form of cryo-injury above and beyond that seen occurring due to classical causes of cell damage (osmotic toxicity and ice formation) during cryopreservation. In spite of this, the permeating CPA should be chosen firstly by their permeating property, and secondly on the basis of their potential toxicity. Because the permeating CPA is responsible for the toxicity (the key limiting factor in cryobiology), different cryoprotectants have been tested for their relative toxicity, and the results indicate that ethylene glycol (EG; MW 62.02) is the least toxic followed by glycerol. Additionally, these highly permeating cryoprotectants are also more likely to diffuse out of the cells rapidly and the cells regained their original volume more quickly upon warming, thus preventing osmotic injury. Therefore, the most common and accepted cryoprotectant for vitrification procedures is ethylene glycol (EG). Today EG is more commonly used in an equimolar mixture with DMSO. Often additives are added to the vitrification solution such as disaccharides. Disaccharides, for example sucrose, do not penetrate the cell membrane, but they help to draw out more water from cells by osmosis, and therefore lessen the exposure time of the cells to the toxic effects of the cryoprotectants. The non-permeating sucrose also acts as an osmotic buffer to reduce the osmotic shock that might otherwise result from the dilution of the cryoprotectant after cryostorage. In addition, permeating agents are able to compound with intracellular water and therefore water is very slowly removed from the cell. Hence the critical intracellular salt concentration is reached at a lower temperature. Removal of the cryoprotectant agent during warming can present a very real problem in terms of trying to reduce toxicity to the cells. Firstly, because of the toxicity of the vitrification solutions, quick dilution of them after warming is necessary; and secondly, during dilution water permeates more rapidly in to the cell than the cryoprotective additive diffuses out. As a consequence of the excess water inflow the cells are threatened by injury from osmotic swelling. In this situation the non-permeating sucrose acts as an osmotic buffer to reduce the osmotic shock. During warming using a high extracellular concentration of sucrose (e.g., 1.0M) counterbalances the high concentration of the cryoprotectant agents in the cell, as it reduces the difference in osmolarity between the intra-and extracellular compartments. The high sucrose concentration cannot totally prevent the cell from swelling, but it can reduce the speed and magnitude of swelling [25, 26, 27].