Microbehavior and heat transfer during freezing of biological substances

在冷冻过程中，细胞内外结冰、渗透水透过细胞膜、细胞变形等行为发生在微观尺度上，对细胞造成严重的损伤，关系到活细胞的生死。这些微观行为发生在至少细胞尺寸水平上，似乎是由于生物材料的物理化学复杂性。水在化学上被限制在水溶液和胶体溶液中，而在物理上被限制在小的空间中或被交联的网络结构所分隔。首先，提出了简化的物理化学模型，讨论了生物细胞冷冻过程中渗透水透过细胞膜等微尺度行为与温度的关系考虑到冷冻保护剂的作用。扩展所提出的细胞单元简化模型，分析了生物物质的冷冻过程， ...更多信息 为进一步发展低温保存技术奠定基础，并进行冻融实验，探讨冻融过程中生物细胞的微观行为与损伤的关系。作为生物细胞的样品，选择性地使用从培养的小麦细胞分离的原生质体。作为使用控制冷却和升温速度的冷台的显微镜观察的结果，由于渗透压差引起的水流入的细胞恢复和细胞内冰的融合随着升温速度而澄清。检测到细胞破裂和肿胀等解冻损伤，发现作用于细胞膜的渗透应力导致这些损伤。细胞的存活也通过使用伊文思蓝的染料排斥试验来检查。结果表明，快速升温对缓慢冻结的细胞危害更大，但对快速冻结的细胞也有好处，其中形成了小冰。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