Isolation of liver cells with Ca2+ and K+ chelating agents. Biochemistry and cell morphology

Abstract

Cell morphology, glutamic pyruvic (GTP) and glutamic oxalacetic transaminases (GOT) concentrations, and the ability to produce glucose or urea from different substrates (pyruvate, alanine, fructose, lactate and glutamine) were studied in isolated mouse and rat liver cells in the presence of Ca2+ and K+ chelating agents (0.1 M sodium perchlorate and 0.027 M sodium citrate with 1 mg/ml bovine albumin; ionic strength: 0.198, pH: 7.4). The chelating agent is perfused through the portal vein of an in situ liver, at low pressure (8 ml/min) at 20 C for 15 min. Cell dispersion is obtained by cutting liver lobes and "massaging" the tissue with a plastic spatula. Wash and cell concentration may be obtained by sedimentation or centrifugation in Krebs III, glucose 150 mg %, improved with 0.16 M pyruvate, 0.1 M fumarate and 0.16 M glutamate. This procedure furnished 53.06 +/- 3.33 X 10(6) cells, which was highly significant (p less than 0.001) with respect to saline controls: 6.11 +/- 1.91 X 10(6). After staining with Papanicolaou, hematoxylin-eosin, and PAS, the cellular material obtained was classified optically into: normal isolated parenchymal liver cells, hepatocyte clumps, "burst" cells, normal blood or reticuloendothelial cells, cellular debris and non-cellular material. Cell morphology showed that a constant perfusion (8 ml/min) with a minimal mechanical treatment, 82.5% of the liver cells appears normal. Biochemical study showed that transaminases are indeed lost, but this loss is below the amount capable of effecting metabolic blockade (3/4 of transaminases remain in liver cells; GOT in cells: 692 +/- 218; GPT in cells. 264 +/- 94; GOT in supernatant: 152 +/- 29; GPT in supernatant: 79 +/- 12 mUI/10(6) cells, after recovering 60 min at 37 C) (means +/- SEM). Conversion of substrates (sodium pyruvate 10 mM, 20 mM D-L alanine, 10 mM fructose and 20 mM D-L sodium lactate) into glucose was statistically significant with respect to the baseline when the liver cells were isolated and recovered (rat liver cells, basal: 25.37 +/- 3.73; pyruvate: 54.04 +/- 7.98; DL-alanine: 62 +/- 10.07; fructose: 264.67 +/- 20.51; DL-lactate: 78.05 +/- 17.99 mmoles/10(6) cels, means +/- SEM). Urea production from 5 mM DL-glutamine was statistically highly significant to the basal with rat liver cell isolated and recovered (basal: 160.60 +/- 3.76; DL-glutamine: 608.47 +/- 16.15 mmoles/10(6) cells; means +/- SEM). The results obtained suggest that liver cells isolated with Ca2+ and K+ chelating agents used as described above are of value for biochemical studies.