Time-Series Recrystallization of Ice Crystals during Preserving and Warming of Frozen Biological Tissues

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Freezing of biological materials is a most fundamental phenomenon in cryosurgery and cryopreservation. Investigation of microscopic behavior of ice crystals and cells during the freezing and thawing is of great importance in relation to the mechanisms of freezing-infuries of cells and protection of cells due to cryoprotectants. The ice crystals growing in the tissues during the freezing causes the mechanical action on cells and the concentration of electrolyte in the unfrozen solution included in the tissues, respectively, resulting in mechanical damage and chemical damage to the tissues. Also, fine ice crystals coarsen selectively in the tissues during the preserving and the warming, which is called recrystallization. The coarse ice crystals increase the mechanical damage. Therefore, understanding of the recrystallization of ice crystals in the tissues is important for the process optimization.Behavior of ice crystals and cells in the biological tissues during the preserving at low te … More mperatures and the warming after rapid-cooling was investigated microscopically in time-series using a confocal laser scanning microscope with a fluorescent dye, acridine orange. Fresh white meat of chicken (2nd pectoral muscles) was used as experimental materials. Attention was paid on the recrystallization of intracellular ice. Influence of warming rate, preserving temperature, and addition of cryoprotectant on the recrystallization characteristics was investigated in the following protocols: 1) warming of rapidly frozen tissues including 2.0M dimethyl sulfoxide (DMSO) (warming rate 0.1℃/min, 1.0℃/min), 2) warming of rapidly frozen tissues without DMSO (warming rate0.1℃/min, 1.0℃/min), 3) constant temperature storage of rapidly frozen tissues including 2.0M DMSO (storage temperature-14℃, -17℃, -19℃), and 4) constant temperature storage of rapidly frozen tissues without DMSO (storage temperature-5℃, -14℃). Size and number of the ice crystals were measured from the image-data of ice crystals and statistically analyzed to obtain frequency, average, and standard deviation of the size (equivalent diameter) of ice crystals, total amount and number density of ice crystals, etc. in time-series during the warming.During the warming, number density of ice crystals decreased monotonously. Frequency of ice crystals with smaller size decreased and that with larger size increased. Due to the change of frequency the averaged size of ice crystals increased up to a maximum value with an increase in temperature, and then due to the dominant effect of melting the averaged size decreased. Total amount of ice crystals increased asymptotically toward the value at thermal equilibrium at the initial stage of recrystallization, and then decreased nearly along the state of thermal equilibrium. The slower-warming caused the more active recrystallization. In the tissues with DMSO, recrystallization proceeded over the wider range of temperature according to the liquidus. During the constant temperature storage, total amount of ice crystals increased asymptotically toward the value at thermal equilibrium. The averaged size of ice crystals continued to increase with time. Addition of DMSO caused the recrystallization in the wider range of low temperature. Less