Active Enhancement of Microscale Heat and Mass Transport in the Process of Cryopreservation and Establishment of the Method of Viability Assessment

要避免细胞内外形成的冰晶造成的致命损害，才能使冷冻保存成功。为此，应适当控制冷冻过程的冷却速度，选择有效的冷冻保护剂抑制冰晶的形成和生长。在低温保存过程中，影响细胞存活的过程有：(1)渗透应力（细胞过度膨胀或收缩），(2)细胞内冻结，(3)细胞外形成冰晶造成的机械应力，(4)生化毒性。一个理想的注册会计师必须能够避免上述所有的压力。本研究[主题1]的目标之一是选择能够避免所有应力(1)~(4)的CPA，即使在缓慢冷却速率下也能实现玻璃化（凝固而不形成冰晶），从而抑制细胞内冻结造成的损伤。一种利用电场的主动技术…更多的电穿孔似乎很有希望。本研究的另一个目的[主题2]是建立细胞在冷冻保存过程中和之后的活力评估方法。我们已经开发了测量细胞和组织力学性能的方法。利用荧光效应对细胞内ATP进行了定量分析。研究结果总结如下。主题1：为了选择符合上述标准的CPA，除了常规使用的甘油和DMSO外，我们还对海藻酸和海藻糖进行了实验。实验结果发现，褐藻酸在低浓度下也具有维持细胞膜形态的功能，因为它的黏度很高，海藻糖在细胞外产生冰晶，从而有效抑制细胞内冻结。此外，将高浓度的CPA引入细胞也能获得令人满意的结果，但还需要更多的实验证据。主题2：为了建立通过测量小尺度标本的压痛来评估生物组织活力的方法，我们开发了一种利用超声脉冲获得组织力学性能的方法。同时，利用细胞发出的荧光来测定细胞中剩余ATP的数量，从而获得细胞的存活率。少

The fatal damage caused by ice crystals formed inside and/or outside the cells should be avoided to lead cryopreservation to success. For this purpose the rate of cooling for freezing process should be controlled properly, and the formation and growth of ice crystals should be suppressed by choosing effective cryoprotective agents (CPA).Among the processes during cryopreservation that govern the survival of cells are ; (1)osmotic stress (excess expansion or shrinkage of cell), (2)intracellular freezing, (3)mechanical stress due to ice crystals formed outside the cell, and (4)biochemical toxicity. An ideal CPA must be capable of avoiding all of those stresses listed above.One of the objectives of this research [Theme 1] is to select CPA that can avoid all the stresses (1)〜(4), and suppress the damage due to intracellular freezing by realizing vitrification (solidification without ice crystal formation) even under slow cooling rate. An active technique utilizing an electric field called … More electroporation seems promising.Another objective of this research [Theme 2] is to establish the means for viability assessment of cells during and after the process of cryopreservation. We have developed the method of measuring mechanical properties of cells and tissues. Also we have carried out the quantitative analysis of ATP in the cells utilizing fluorescence effect. The summary of the research results is as follows.Theme 1 : In order to select the CPA that may satisfy the criteria described above, we have carried out experiment with alginic acid and trehalose in addition to glycerol and DMSO employed conventionally. As the result of experiment, it has been found that alginic acid has a function of maintaining the form of cell membrane because of the high viscosity even under low concentration, and trehalose generates ice crystals outside the cell, thus being effective in suppressing intracellular freezing. In addition, introduction of CPA with high concentration into cells has been found to lead to satisfying result, though much more exprimental evidence is needed.Theme 2 : In order to establish the means of assessing viability of biological tissue by measuring tenderness of small-scale specimen, we have developed a method of obtaining the mechanical properties of the tissue using ultrasonic pulses. At the same time, the ratio of survival of cells has been obtained by determining the quantity of ATP remaining in the cell making use of fluorescent light emitted from them. Less