Effects of temperature and freezing on hepatocytes isolated from a freeze-tolerant frog

Metabolically active hepatocytes prepared from freeze-tolerant wood frogs, Rana sylvatica, were used to examine the direct effects of temperature and freezing on cryoprotectant synthesis and to assess the effectiveness of the natural cryoprotectant glucose in the freezing preservation of the isolated cells. Freshly isolated hepatocytes showed slow leakage of lactate dehydrogenase, readily synthesized urea, and oxidized a variety of 14C-labeled substrates. Effects of temperature on glucose production by isolated hepatocytes showed a normal Arrhenius relationship. However, compared with 0 degrees C control cells, either incubation at higher temperatures or freezing at -3 degrees C reduced the activity of glycogen phosphorylase alpha. These data suggest that the freezing-induced cryoprotectant production that occurs in vivo is not due to direct action of either low temperature or freezing on liver cell metabolism. The natural cryoprotectant glucose was also an excellent cryoprotectant of hepatocytes in vitro. In the absence of glucose, freezing caused a substantial leakage of lactate dehydrogenase from isolated hepatocytes, the rate of leakage increasing as freezing temperature decreased. Addition of 200-600 mM glucose to the incubation medium (similar to natural levels) fully protected cells against damage during freezing at -4 or -8 degrees C, normal freezing temperatures experienced by these frogs. Glucose also greatly improved freezing survival of isolated frog hepatocytes at ultralow temperatures (-80 or -196 degrees C).