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

为使冷冻保存成功，应避免细胞内外冰晶造成的致命性损伤。为此，应适当控制冷冻过程的降温速度，通过选择有效的冷冻保护剂(CPA)来抑制冰晶的形成和生长。在低温保存过程中，控制细胞存活的过程包括：(1)渗透胁迫(细胞过度膨胀或收缩)，(2)细胞内冻结，(3)细胞外冰晶形成的机械应力，(4)生化毒性。理想的CPA必须能够避免上述所有应力。本研究的目标之一[主题1]是选择能够避免所有应力(1)~(4)的CPA，并通过实现玻璃化(固化而不形成冰晶)来抑制细胞内冻结造成的损害，即使在缓慢的冷却速度下也是如此。一种利用称为…的电场的有源技术更多的电穿孔似乎是有希望的。这项研究的另一个目标[主题2]是建立在低温保存过程中和之后对细胞活力进行评估的方法。我们开发了测量细胞和组织的机械性能的方法。并利用荧光效应对细胞内的三磷酸腺苷进行了定量分析。研究结果总结如下。研究内容1：为了筛选出符合上述标准的CPA，我们在常规使用的甘油和DMSO的基础上，进行了海藻酸和海藻糖的实验。实验结果发现，海藻酸即使在低浓度下也因其高粘度而具有保持细胞膜形态的功能，海藻糖在细胞外产生冰晶，从而有效地抑制细胞内的冻结。此外，将高浓度的CPA引入细胞内也取得了令人满意的结果，尽管还需要更多的实验证据。主题2：为了建立通过测量小尺寸标本的嫩度来评估生物组织活力的手段，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测定细胞内剩余的三磷酸腺苷的量，从而得到细胞的存活率。较少

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