Controlled rate cooling

Abstract

Controlled rate cooling is based on osmotic regulation of cell contents and freeze-induced dehydration. The samples are pretreated in cryoprotectant solutions and cooled at a standard rate to an intermediate temperature such as -35°C or -40°C, with ice nucleation initiated at about -9°C. At the freezing point of the cryoprotectant solution, ice nucleation is initiated, and ice forms in the cryoprotectant solution and the intercellular spaces. The cytoplasm remains unfrozen due to solute concentration and the cell wall protects the cell membrane from damaging ice crystals. As the temperature is further decreased to -35°C or -40°C, the extracellular solution becomes increasingly icy and the intracellular solutes become highly concentrated. The plant cells lose water to the exterior ice and the cytoplasm is further concentrated. The intracellular freezable water is safely reduced before samples are plunged into liquid nitrogen (LN). If the cells are optimally dehydrated, the cytoplasm vitrifies on contact with LN. If the samples are under dehydrated, leaving freezable water in the cytoplasm, ice will form. If over dehydrated, the cells may die from desiccation. Controlled rate cooling is very efficient for storing suspension and callus cultures, embryogenic cultures, and in-vitro shoot tips from temperate and subtropical plants. The advantages of controlled rate cooling include the use of standardized procedures, programmed cooling rates, and large batch sizes, and the effective use of technician time.