Cryopreservation of tissues and organs, including artificial organs, could be one of the important steps in the medical service that brings the progress in the tissue engineering to realization. In this case, high viability of cryopreserved cells is critical to recovery after transplantation. In contrast, in the cryosurgery, which is expected to expand its application as a minimally invasive treatment of cancer, malignant cells should be destructed completely to prevent from recurrence. The appropriate freeze-thaw protocol is therefore needed to be established for cryopreservation or cryosurgery depending on specific type of tissues and organs. Although it is determined empirically, the underlying mechanism of cell injury by freezing has been explored for a long time to give a scientific basis of the process. The experiments with a cell suspension showed that the cell injury during slow freezing to a relatively higher sub-zero temperature was attributed to the mechanical stress from the extracellular ice, while the effect of elevated concentration of solutes became more crucial to cell damage at lower temperatures [1]. However, there are some studies that indicates the difference in the freeze tolerance between cell suspensions and attached monolayers, some of which indicated higher susceptibility of monolayers to freezing than cell suspension [2] and the other suggested reverse [3,4]. The goal of our study is thus to validate the difference in freezing injury between isolated cells and tissues that are more important in aforementioned applications and clarify the mechanism. We used cells adhered to a surface as a first simple model of cells in tissues. The cells adhered on a surface at low number density were used to highlight the effect of cell-to-surface interaction without cell-to-cell interactions. In the present study we first demonstrate that the survival of cells adhered on a surface is lower than those in the suspension after a freeze-thaw manipulation. Then the osmotic response to concentration increase was examined to clarify if the extent of dehydration is different between these two types of cells. The cells were observed by a laser confocal scanning microscope that allows real-time 3-D observation.

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