Background. Various cryopreservation techniques have been investigated to elongate preservation time, however, most have failed to be clinically induced because of damage due to ice crystal formation. Subzero nonfreezing conditions could theoretically reduce organ metabolism without damage due to ice crystal formation. We evaluated the superiority of subzero nonfreezing storage compared with conventional hypothermic storage using isolated rat hepatocytes stored in University of Wisconsin (UW) solution without cryoprotectants. Methods. Hepatocytes of Wistar rats isolated by collagenase digestion were suspended in UW solution and divided into the following three groups: subzero nonfreezing group (-4°C), zero nonfreezing group (0°C), and control group (4°C). They were stored for 48 hr at the temperatures indicated. After 24 and 48 hr of storage, we carried out a trypan blue exclusion test and a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and measured lactate dehydrogenase release, lactic acid, ATP content, and the ability of hepatocytes to synthesize urea. After 48 hr of storage, morphological differences between the control group and the subzero nonfreezing group were investigated by scanning and transmission electron microscopy. Results. Significant improvements of the trypan blue exclusion test and ATP contents in the subzero nonfreezing group were observed. Lactic acid production was also significantly suppressed in the subzero nonfreezing group compared with that in the control group. The MTT assay value was significantly better at -4°C than at 4°C. The rate of urea synthesis at -4°C was higher than that at 4°C. Electron microscopy revealed that subzero nonfreezing delayed the lethal bleb-forming process of stored hepatocytes, which was followed by mitochondrial swelling, compared with the control group. Conclusions. Subzero nonfreezing storage (-4°C) in UW solution could provide better preservability for isolated rat hepatocytes with protection against hypoxic cell injury compared with conventional hypothermic storage (4°C).
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