Microbehavior and heat transfer during freezing of biological substances

在冷冻过程中，细胞外和细胞内的结冰、水渗透细胞膜、细胞变形等行为发生在微观尺度上，对活细胞造成严重伤害，关系到活细胞的生死。这些微观行为至少发生在细胞大小水平上，似乎是由于生物材料的物理化学复杂性。水在化学上受限于水溶液和胶体的状态，而后者在物理上被限制在小空间或被交联的网络结构分隔开。首先，提出了简化的物理化学模型，并考虑了冷冻保护剂的作用，讨论了生物细胞冷冻过程中细胞外和细胞内结冰、细胞膜渗透水等微尺度行为随温度的变化规律。在细胞元素简化模型的基础上，进一步分析了生物物质的冷冻过程，为进一步发展冷冻保存技术提供了基础知识。此外，还通过冻融实验探讨了生物细胞的微观行为与冻融损伤之间的关系。作为生物细胞的样本，从培养的小麦细胞中分离的原生质体被选择性地使用。通过控制冷暖速度的冷段微观观察，澄清了由于渗透压差引起的水流入对细胞的恢复，以及细胞内冰的融合。检测到细胞的破裂、膨胀等融化损伤，发现这些损伤是由细胞膜上的渗透应力引起的。采用Evans蓝染色排除法检测细胞存活率。结果表明，快速升温对慢冻细胞的危害更大，但对形成小冰的快冻细胞有益。少

During freezing, the extra-and intra-cellular ice formation, osmotic water permeation through cell membrane, deformation of cell and other behaviors occur at microscale and they bring serious injuries connecting to the life-and-death of living cells. These micro behaviors occurred on at least cell size level seem to be due to the physicochemical complexity of biological materials. Water is chemically restrained in the states of aqueous solution and colloidal solution, and those are physically confined in small spaces or compartmentalized by the cross-linked network structures.Firstly, simplified physicochemical model was proposed and the extra- and intra-cellular ice formation, osmotic water permeation through cell membrane and other microscale behavior durig the freezing of biological cell were discussed as a function of temperature considering the effect of cryoprotectant. Extending the simplified model proposed for the cell element, the freezing of the biological substances was anal … More yzed to get basic understanding for developing the cryopreservation technique.Furthermore, the freezing and thawing experiments were conducted to pursue the relation between the micro-behavior of biological cell and the injury during freezing and thawing. As a sample of biological cells, protoplasts isolated from cultured wheat cells were selectively used. As the results of microscopic observation using a cold stage whose cooling and warming velocities were controlled, the recovery of cell by water influx due to osmotic pressure difference, and the fusion of intracellular ice were clarified with warming velocity. The thawing injuries such as rupture and swell of the cells were detected, and it was found that the osmotic stress acting on the cell membrane causes these injuries. The survival of cells were also inspected by dye-exclusion test using Evans Blue. The results suggested the rapid temperature-rising is more harmful for slow-freezed cell, but also useful for rapid-freezed cell in which small ice formed. Less