Abstract
Isolated adult rat hepatocytes were prepared under hypoxic conditions, following a two-step procedure as described by Seglen. A simple recirculating apparatus was used and some modifications were made. Electron microscope studies revealed that the fine structure of the hepatocytes was not extensively damaged during cell preparation. Isolated hepatocytes prepared under hypoxic conditions are functional as demonstrated by their ability to carry out gluconeogenesis under normal and stimulated conditions, by the selectivity of the cell membrane towards various substrates and by the preservation of insulin receptors on the cell membrane. As previously observed for other cell types, survival of hepatocytes after freezing was dependent on the cooling rate. About 80% of cells appeared to survive when cooled at 7-2 °C/min; this apparent optimum cooling rate was also found suitable to preserve insulin-binding sites; on the other hand, the gluconeogenic capacity of hepatocytes frozen under these conditions was consistently decreased and the fine structure of organelles was damaged.
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