Cultured primary turtle hepatocytes: a cellular model for the study of temperature and anoxia.

Turtle hepatocytes are a nonexcitable model for metabolic depression during low-temperature and/or anoxic overwintering conditions. Cytoskeletal structure and mitochondrial distribution are continuously modified in cells, and we hypothesized that metabolic depression would inhibit such processes as cell attachment and spreading and promote withdrawal of cell protrusions and peripheral mitochondria. After developing a methodology for culturing painted turtle hepatocytes, two-dimensional (2-D) area and maintenance of cell attachment after a media change were used as indicators of structural rearrangement and spreading/volume. These were measured after incubating cells at varying temperatures and with or without the inclusion of cyanide (chemical proxy for anoxia). Experiments were performed using cells from 22°C- or 5°C-acclimated turtles. Live-cell imaging was used to monitor the effect of cyanide exposure on the distribution of mitochondria. We also acclimated cultured cells from 22°C-acclimated turtles to 4°C in vitro and scored withdrawal of protrusions. Only cells isolated from 5°C-acclimated turtles and incubated at 4°C had reduced attachment to fibronectin substrate, but cyanide exposure had no effect. These cells also had a 30% smaller 2-D area than those from 22°C-acclimated turtles. There was no change in mitochondrial distribution during cyanide perfusion. Finally, 4°C acclimation in vitro resulted in the withdrawal of protrusions over 14 days. Taken together with the results from cells acclimated to low temperature in vivo, this suggests inhibition of structural rearrangement and protrusion stability by low temperature acclimation, but not cyanide exposure. Our cultured primary hepatocyte system will facilitate further study of the role of structural dynamics in reversible metabolic depression. We have optimized a methodology for two-dimensional (2-D) culturing of primary western painted turtle hepatocytes and used this model to study the effects of cyanide and temperature on structural rearrangement, and the effect of cyanide on mitochondrial distribution. Our results suggest that low temperature acclimation, either in vivo or in vitro, inhibits cell protrusions and structural rearrangement. Acute cyanide exposure did not inhibit structural rearrangement or alter mitochondrial distribution.