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Abstract

The first technique for large-scale preparation of isolated hepatocytes was described in 1953 and involved perfusion of rat liver under pressure with a Ca(2+)-free solution containing a chelating agent. Various modifications of this technique were in use over the next ten years, until it was demonstrated that cells prepared in this manner were grossly damaged, losing most of their cytoplasmic enzymes during the preparative procedure. The successful preparation of intact isolated hepatocytes by collagenase-treatment of liver was achieved in 1967, and the widespread use of intact hepatocyte suspensions was accelerated by the development soon after of high-yield preparative techniques involving perfusion of the liver with a medium containing collagenase. The introduction of the isolated hepatocyte preparation has enabled experimental studies that otherwise would not be feasible. Important advances have been the use of cultured hepatocytes, frequently of human origin, for the investigation of the metabolism and toxicology of potential therapeutic agents. Success in this field has been achieved through the steady improvement in techniques for the maintenance in culture of differentiated hepatocytes, and in particular their cytochrome P450 complexes. Another area showing considerable promise is the employment of hepatocytes, generally from a porcine source, in temporary support systems for patients with acute liver failure. Our own studies have concentrated on the demonstration of long-range interactions between hepatocyte compartments which suggest that energy transfer between cell compartments can take place without ATP turnover.

Thioredoxin-interacting Protein (Txnip) Is a Critical Regulator of Hepatic Glucose Production

“…Hepatocyte Isolation and Culture-Mouse hepatocytes were isolated by a modified two-step perfusion procedure (37,38). In brief, mice were perfused retrograde through the hepatic vein via the inferior vena cava and out the severed portal vein, with the inferior vena cava clamped above the diaphragm.…”

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

“…In the first step, the perfusion of the liver piece with the EGTA-containing perfusion solution causes a depletion of Ca 2+ within the tissue. As a consequence, the desmosomes, which are responsible for cell–cell connections, undergo structural changes that cause a weakening or an irreversible complete loss of cell–cell linkages, so that the desmosomes are unable to reform even when the tissue is perfused with a second perfusion solution containing high concentrations of Ca 2+ , which is needed for the collagenase activity (Berry et al 1997). During step 2 of the perfusion process, cell–matrix contacts are destroyed by the digestion of ECM proteins with collagenase.…”

“…Low collagenase activity and high protease/trypsin activity correlate with a decrease in isolation efficiency with regard to cell yield and cell viability (Berry et al 1997). In general, but not invariably, the most rapid digestions and the best yields of intact cells are achieved with preparations of highly active collagenase (Berry et al 1997).…”

Structure, function and regulation of pyruvate carboxylase

“…Several studies have indicated that the glycolytic and gluconeogenic enzymes present in the cell are associated in the form of multienzyme complexes [56,57]. PC is also found to be specifically associated with other mitochondrial enzymes : in binary complexes with mitochondrial aspartate transferase or malate dehydrogenase, in a ternary complex with aspartate aminotransferase and glutamate dehydrogenase, and in a quaternary complex with aspartate aminotransferase, glutamate dehydrogenase and malate dehydrogenase [58].…”