Selective isolation of intact periportal or perivenous hepatocytes by antero‐ or retrograde collagenase gradient perfusion

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Abstract
ABSTRACT— To study the role of functional heterogeneity in the pathogenesis of liver damage, a new technique for separation of intact periportal and perivenous parenchymal cells was developed. After a brief portal perfusion in situ, the upper vena cava was also cannulated and the medium was simultaneously and slowly pumped via the portal and hepatic veins so that the medium had to ooze through the liver capsule. For the isolation of periportal cells collagenase was added selectively to the medium perfused through the portal vein in order to concentrate the digestive effects of collagenase in the periportal parenchyma. Conversely, to obtain cells of perivenous origin, collagenase was added to the medium perfused via hepatic veins. After 6–7 min and a brief reoxygenation by portal perfusion the liver was rapidly sliced. The slices were shaken for 3 min in preoxygenated medium and free hepatocytes were isolated by centrifugation. Injured cells were eliminated by brief and gentle trypsin‐DNase treatment terminated with trypsin inhibitor. With this procedure, 0.2–1 g packed cells with viability of over 90% could be routinely obtained. LDH‐leakage during incubation of either hepatocyte preparation was small and similar to that of hepatocytes isolated by conventional collagenase perfusion. Histological HE‐stained preparations of liver parenchyma after perfusion demonstrated selective loosening of either periportal or perivenous cellular subpopulations. Moreover, the activity of alanine aminotransferase was three times higher (p<0.001) in “periportal” than in “perivenous” cells. This difference is similar to that obtained by the microdissection technique. The isolated “perivenous” cells were significantly larger (+28%, p<0.001) than “periportal” cells, as measured from the packed cell volume. The results demonstrate that with this technique intact hepatocytes of either periportal or perivenous origin can be obtained in sufficient amounts to enable their further characterization by incubation or culture experiments.