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ABSTRACT: 1.1. A simple procedure for the isolation of rat liver parenchymal cells, modified from the collagenase-hyalutonidase method of Berry and Friend, is described in detail.2.2. It involves a flow-through in situ perfussion of the rat liver at physiological flow rate and pressure, using enzymes and EDTA (2 mM) in saline medium, with or without the addition of 20% washed bovine erthrocytes.3.3. The cell preparation was studied with respect to the following biochemical characteristics: Hexokinase and glucokinase activity; respiration rate in saline medium with and without substrate; permebility to malate, glutamate, 3H2O and [14C]inulin; glucose- and glycogen-synthesizing capacities; adenine nucleotide and K+ contents.4.4. The effects on these parameters of the omission of eruthrocytes from the perfusion media, and thereby a low oxygenation of the liver during the perfusion, was examined.5.5. Cells prepared with or without erythrocytes showed no differences in respiration rate, rate of gluconeogenesis of permeability to malate and glutamate. The cells were able to maintain a gradient of malate and glutamate comparable to that in the isolated perfused rat liver. The 3H2O/[14C]inulin ratio indicated some cell destruction ort penetration of inulin into the cells, independent of the isolation method used.6.6. Cells prepared with erythrocytes, however, showed a four to seven times higher rate of glycogen synthesis and double the adenine nucleotide content as compared with those prepared without erythrocytes in the perfusion media.7.7. Compared to the method of Berry and Friend, the method described here is simpler in perfusion procedure and uses physiological flow rate and pressure. The isolated cells recover from a loss of K+ to return to near normal values (90 μmoles/ml parked cells) as opposed to the cells of the former method, which show a 60% irreversible loss of the ion.8.8. The results are discussed in view of earlier stduies on the isolation of hepatocytes, and a comparison is drawn with the characteristics of the isolated perfused rat liver and rat liver slices.
Biochimica et Biophysica Acta (BBA) - General Subjects.
