A simple and rapid method of dissociating hepatocytes from fixed liver of the mouse

A simple and rapid method of dissociating hepatocytes of fixed liver tissue is described. Mouse liver was fixed by vascular perfusion with sodium phosphate buffered 2% formaldehyde-2% glutaraldehyde solution containing 0.02% picric acid and then osmicated in 2% OsO4 in phosphate buffer by immersion. Hepatocytes are easily dissociated by tapping the fixed tissue blocks in distilled water with a glass rod or by ultrasonics. This method results in very low cell fragility and a high yield of well preserved hepatocytes in suspension. For light microscopic examination the separated cells may be uniformly spread on a slide glass coated with Mayer's egg albumen and stained. Electron microscopic evaluation of the dispersed cells indicated that they have intact cell membranes and retain the integrity of their cytoplasm and nuclei well. This method is most suitable for accurate determination of the nuclear content and size of individual liver cells, as well as of the number of mitotic cells, and is potentially useful for gathering other information on the morphometric cytology of the liver.