Improved isolation, separation and cytochemistry of living cells.
1. This paper describes improved methods of obtaining, purifying and studying bulk suspensions of isolated living hepatocytes and other cells of adult rats and urodeles. 2. The cells were isolated largely by dissolving the hepatic ground substance through the extracorporeal portal perfusion and further incubation of the excised liver with 0.05% collagenase and 0.1% hyaluronidase. The different kinds of cells were then separated from one another by counter-current centrifugation. The isolated cells were examined by differential interference, phase-contrast, amplitude-contrast, ultraviolet, fluorescence and electron microscopy. Various cytochemical tests were carried out. Whenever possible, for each method of examination, the isolated cells were compared with cells of the same kind which had not undergone isolation. 3. Dye-exclusion, lysochromy, fluorescence and differential interference microscopical analysis indicated viability rates between 75 and 99%. Succinate dehydrogenase activity was preserved at a high level in nearly all isolated cells. In hepatocytes, the essentially extracellular cells. In hepatocytes, the essentially extracellular 'soluble' alkaline phosphatase activity of bile canaliculi was retained. Living hepatocytes were studied by super-modulating methods of microscopy for the first time, with somewhat unexpected findings. It now seems probable that previous methods of tissue preparation produced gross alterations in hepatocyte mitochondria. The assessment of the viability of isolated cells was re-examined. 4. The methods described may permit a more meaningful correlation between biochemical, cytochemical, ultrastructural and biophysical findings than that obtainable by the use of current methods.