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

生物材料的冻结是低温外科和低温保存中最基本的现象。研究冰晶和细胞在冻结和解冻过程中的微观行为，对于研究细胞的冻胀机制和冷冻保护剂对细胞的保护具有重要意义。冰冻过程中在组织中生长的冰晶分别对细胞产生机械作用，使组织中含有的未冷冻溶液中的电解液浓度升高，从而对组织造成机械损伤和化学损伤。此外，在保存和升温过程中，细小的冰晶在组织中选择性地粗化，这被称为再结晶。粗大的冰晶增加了机械损伤。因此，了解冰晶在组织中的重结晶过程对工艺优化具有重要意义。冰晶和生物组织细胞在低TE…保存过程中的行为用共聚焦激光扫描显微镜观察了更多的温度和快速冷却后的升温，并用荧光染料丫啶橙进行了观察。以新鲜鸡肉白肉(第二胸肌)为实验材料。对细胞内冰的重结晶进行了研究。研究了升温速度、保存温度和添加冷冻保护剂对重结晶特性的影响：1)包括2.0M二甲基亚砜的快速冷冻组织的升温(升温速率分别为0.1Mmin、1.0Min/℃)；2)快速冷冻组织的非二甲基亚砜的升温(升温速率分别为0.1min、1.0min)；3)快速冷冻组织的恒温保存，包括2.0M的二甲基亚砜(保存温度为-14℃、-17℃、-19℃)；(4)不含二甲基亚砜的快速冷冻组织恒温保存(保存温度-5℃，-14℃)。从冰晶图像数据中测量冰晶的大小和数量，并对其进行统计分析，得到变暖过程中冰晶大小(当量直径)、冰晶总数和数密度等在时间序列上的频数、平均值和标准差等。较小尺寸的冰晶出现的频率降低，较大尺寸的冰晶出现的频率增加。由于频率的变化，冰晶的平均尺寸随着温度的升高而增大到一个最大值，然后由于融化的主导作用，平均尺寸减小。在再结晶初期，冰晶总量向热平衡值逐渐增加，然后几乎沿热平衡状态逐渐减少。升温速度越慢，再结晶越活跃。在含有DMSO的组织中，再结晶是在较宽的温度范围内进行的。在恒温储存过程中，冰晶总量向热平衡值渐近增加。冰晶的平均大小随着时间的推移而不断增大。加入DMSO后，在较宽的低温范围内发生再结晶。较少

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