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

冷冻生物组织保存和加温过程中冰晶的时间序列再结晶

• 批准号: 13650209
• 负责人: ISHIGURO Hiroshi
• 金额: $ 2.18万
• 依托单位: Kyushu Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13650209/
• 关键词: Frozen Biological Tissues; Cryoprotectant; Preservation Process; Warming Process; Ice Crystals; Recrystallization; Confocal Laser Scanning Microscope; Time-Series Statistical Characteristics; 生体組織; 再結晶化現象; 急速冷却; 緩速加温; 細胞内氷結晶; 統計的特性; 時系列変化

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

生物材料的冷冻是冷冻外科和冷冻保存中最基本的现象，研究冰晶和细胞在冷冻和解冻过程中的微观行为对于细胞冷冻损伤的机制和冷冻保护剂对细胞的保护具有重要意义。冷冻过程中组织中生长的冰晶分别引起细胞的机械作用和组织中未冷冻溶液中电解质的浓缩，从而导致组织的机械损伤和化学损伤。此外，在保存和升温过程中，细小的冰晶会选择性地在组织中变粗，这被称为重结晶，因此，了解组织中冰晶的重结晶对于过程优化很重要。使用共焦激光扫描显微镜按时间序列对生物组织中冰晶和细胞在低温保存和快速冷却后升温过程中的行为进行了显微研究。采用荧光染料吖啶橙作为实验材料，研究了升温速率、保存温度和冷冻保护剂添加对重结晶特性的影响。在以下方案中：1）快速冷冻组织加温，包括2.0M二甲基亚砜（DMSO）（加温速率0.1℃/min， 1.0℃/min），2）不含DMSO的快速冷冻组织升温（升温速率0.1℃/min，1.0℃/min），3）含2.0M DMSO的快速冷冻组织恒温储存（储存温度-14℃， -17℃，-19℃），4​​）不含DMSO的快速冷冻组织恒温保存（保存温度-5℃，-14℃）冰的大小和数量。根据冰晶的图像数据测量冰晶的晶体，并对冰晶尺寸（当量直径）的频率、平均值和标准偏差、冰晶的总量和数密度等在时间序列上进行统计分析。在变暖过程中，冰晶数量密度单调下降，尺寸较小的冰晶出现频率降低，而尺寸较大的冰晶出现频率增加，由于频率的变化，冰晶的平均尺寸增加至最大值。增加温度升高后，由于融化的主导作用，冰晶总量逐渐向再结晶初始阶段的热平衡值增加，然后沿着升温导致的热平衡状态逐渐减少。在含有DMSO的组织中，根据液相线，重结晶在更宽的温度范围内进行，在恒温储存过程中，冰晶总量增加。冰晶的平均尺寸随着时间的推移逐渐增加，导致在更宽的低温范围内发生重结晶。