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

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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