Effect of Three-Dimensional Cellular Structure on Post-Thaw Viability in the Cryopreservation of Biological Tissue

通过选择细胞密度制备了人工组织、单层培养细胞和悬浮细胞。将它们冷冻作为冷却速率的参数，并评估解冻后的活力。通过观察染色的人工组织和单层培养细胞的相位差光镜，明确了未冷冻状态下的细胞结构。实验采用人成纤维细胞。采用胶原海绵（Koken CS-100，直径20 mm，厚度1 mm）中细胞密度为10^5 ~ 10^7个细胞/cm^3的人工组织，培养皿（直径35 mm）中面积细胞密度为10^4 ~ 10^6个细胞/cm^2的单层细胞，细胞密度为10^5 ~ 10^7个细胞/cm^3的细胞悬浮。冷冻保护剂选用10%的二甲亚砜。采用液氮作为冷却源，通过调节温度控制器（Yokogawa UP-750），将样品从10℃冷冻至低于-180℃，冷却速度为0.1 ~ 100℃/min。将样品解冻；用台盼蓝排斥法测定细胞活力。用相差显微镜观察苏木精-伊红染色的解冻人工组织切片和解冻单层培养细胞。解冻后的细胞悬液中加入荧光染料（Molecular Probes SYTO 13），倒置荧光显微镜（Nikon TE300-DEF-S）检测细胞活力。结果表明，随着人工组织和单层培养细胞细胞密度的增加，最佳冷却速率向低冷却速率侧偏移，细胞活力下降。然后在高密度人工组织和单层培养细胞中观察细胞接触情况。而对于悬浮细胞，细胞密度的增加并没有引起显著的活力下降。球形悬浮细胞比纺锤形单层培养细胞体积小。因此，我们认为悬浮细胞的活力并没有降低，因为即使相同密度的细胞也不会因为球形和小的形状而相互接触。此外，荧光材料测定的活力与台盼蓝测定的活力具有相关性。少

Artificial tissue, a monolayer culture cell and a suspension cell were prepared by the optional cell density. They were frozen as a parameter of a cooling rate, and the post-thaw viability was evaluated. The cellular structure of the un-frozen condition was clarified by observing phase difference light microscope in the dyed artificial tissue and the monolayer culture cell.Human fibioblast was used for the experiment. Artificial tissue cultured with the cell density of 10^5 to 10^7 cells/cm^3 in the collagen sponge (Koken CS-100, 20 mm in diameter and 1 mm in thickness), the monolayer cell with the area cell density of 10^4 to 10^6 cells/cm^2 in culture dish (35 mm in diameter), and cell suspension with the cell density of 10^5 to 10^7 cells/cm^3 were used. Dimethyl sulfoxide of 10% was used for the cryoprotectant. Liquid nitrogen was used for a source of cooling, and a sample was frozen with a cooling rate of 0.1 to 100℃/min from 10 to lower than -180℃ by regulating the voltage suppli … More ed to the heater using a temperature controller (Yokogawa UP-750). The sample was thawed ; the cell viability was evaluated with the trypan blue exclusion assay. A slice of the unfreezing artificial tissue dyed with the hematoxylin-eosin and the un-freezing monolayer culture cell were observed by a phase contrast microscope. Moreover, fluorescent dye (Molecular Probes SYTO 13) was added to the cell suspension after thawing, and the viability was measured by using the inverted fluorescence microscope (Nikon TE300-DEF-S).As a result, with increasing the cell density in the artificial tissue and the monolayer culture cell, the optimal cooling rate shifted to the low cooling rate side, and the viability decreased. Then, contact of the cells was observed in high cell density of artificial tissue and the monolayer culture cell. However, as for the suspension cell, there was no remarkable viability decline caused by increase in cell density. The suspension cell of spherical shape was smaller than the monolayer culture cell of spindle shape.Therefore, it was thought that the viability of a suspension cell did not decrease because even the same cell density did not contact each other due to spherical and small shape. In addition, the viability measured by fluorescent material and by the trypan blue correlated. Less