Hierachical Study on Heat Transfer and Viability of Cell during Freezing of Biological Tissue

冷冻可以减缓或停止一些生物反应，以保存生物组织，解冻可以使其恢复生命。冻结和解冻对生命系统是致命的，这也是事实。在冷冻过程中，细胞外和细胞内的结冰、水渗透细胞膜、细胞变形等行为都发生在微观尺度上，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。本课题首先提出了简化的细胞元件冻结模型，讨论了生物细胞冻结过程中细胞外和细胞内结冰、细胞膜渗透水等微尺度行为与温度的关系。其次，研究了细胞活力与冻融过程中微行为的关系。实验中，对小麦原生质体进行了显微观察，并用荧光法检测细胞活力。冻融过程中遇到的渗透条件也通过渗透操作来实现。综上所述，考虑“溶液效应损伤”和“细胞内冰损伤”，对冷冻过程中的细胞损伤进行了估计。第三，采用传热和热力学模型对生物组织冷冻过程进行了数值模拟。在这些结果的基础上，结合传热过程讨论了生物组织冷冻过程中细胞的活力。

Freezing can slow down or stop some biological reactions for preserving the biological tissue, and thawing can bring it back to life. It is also true that freezing and thawing are lethal to the living system. 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.In this research project, firstly, simplified freezing model of cell element was proposed, and the extra- and intra-cellular ice formation, osmotic water permeation through cell membrane and other microscale behavior during the freezing of biological cell were discussed as a function of temperature.Secondly, viability of the cell was studied in relation with the micro-behaviors during freezing and thawing. In experiments, microscopic observation of wheat protoplasts was performed, and the cell viability was inspected by the fluorescence test. The osmotic conditions encountered during freeze-thaw process were also realized by osmotic manipulations. Summarizing these results, the cell damage during freezing process was estimated taking account of "solution effect injury" and "intracellular ice injury".Thirdly, the freezing process of biological tissue was numerically simulated using heat transfer and thermodynamic model. On the basis of these results, viability of the cell during freezing of biological tissue was discussed in conjunction with the heat transfer process.