Publisher Summary This chapter describes the isolation and use of liver cells. The technique is based on liver perfusion with collagenase after removal of Ca 2+ by preperfusion with a chelator. Moderately high yields of viable, single hepatocytes with a smooth, spherical appearance and essentially free of nonparenchymal cells are obtained. Early attempts to isolate hepatocytes employed mechanical force and, subsequently, perfusion of the liver with Ca 2+- or K+-chelators but were unsuccessful in obtaining viable cells in high yields. This was not achieved until the isolation of rat hepatocytes by the use of the digestive enzymes collagenase and hyaluronidase was introduced by Howard and collaborators. Although most of the techniques recently employed to obtain isolated hepatocytes involve perfusion of the liver with digestive enzymes, attempts are also made to avoid the perfusion step and achieve hepatocyte isolation by incubating cut pieces of the liver in enzyme-containing solutions. The presently available results suggest that isolated hepatocytes—and cell suspensions isolated from certain other target tissues—may be a useful model for drug toxicity studies in the future.

Isolation and use of liver cells.

Antidiabetic plants and their active constituents.

Heterotypic cell interaction between parenchymal cells and nonparenchymal neighbors has been reported to modulate cell growth, migration, and/or differentiation. In both the developing and adult liver, cell-cell interactions are imperative for coordinated organ function. In vitro, cocultivation of hepatocytes and nonparenchymal cells has been used to preserve and modulate the hepatocyte phenotype. We summarize previous studies in this area as well as recent advances in microfabrication that have allowed for more precise control over cell-cell interactions through 'cellular patterning' or 'micropatterning'. Although the precise mechanisms by which nonparenchymal cells modulate the hepatocyte phenotype remain unelucidated, some new insights on the modes of cell signaling, the extent of cell-cell interaction, and the ratio of cell populations are noted. Proposed clinical applications of hepatocyte cocultures, typically extracorporeal bioartificial liver support systems, are reviewed in the context of these new findings. Continued advances in microfabrication and cell culture will allow further study of the role of cell communication in physiological and pathophysiological processes as well as in the development of functional tissue constructs for medical applications.

http://lmrt.mit.edu/sites/default/files/Bhatia1999_FASEBJ_0.pdf

Effect of cell–cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells

Microcystins and nodularin, isolated from toxic blue-green algae, are hepatotoxic monocyclic polypeptides. Both microcystins and nodularin inhibited in vitro protein phosphatase activity present in a cytosolic fraction of mouse liver, bound to the okadaic acid receptors, protein phosphatases 1 and 2A, and thus resulted in the increase of phosphoproteins; this was referred to as the apparent “activation” of protein kinases. Their concentrations causing 50% of the maximal effects are comparable to that of okadaic acid, a potent protein phosphatase inhibitor and a potent tumor promoter, in the nanomolar range of concentration. The increase of phosphoproteins was observed in rat primary cultured hepatocytes and was subsequently associated with morphological changes, which appeared to be a step in the process of hepatotoxicity. The well-known hepatotoxic compounds,α-amanitin and phalloidin, did not show any effects similar to those of microcystins, nodularin and okadaic acid. It is suggested that the hepatotoxicity of microcystins and nodularin may result from inhibition of protein phosphatases and the increase of phosphoproteins.

Inhibition of protein phosphatases by microcystins and nodularin associated with hepatotoxicity.

Aberrant energy metabolism is one characteristic of diabetes mellitus (DM). Two types of DM have been identified, type 1 and type 2. Most of type 2 DM patients eventually become insulin dependent b...

Resveratrol, a red wine antioxidant, possesses an insulin-like effect in streptozotocin-induced diabetic rats

The enzymic isolation of adult rat hepatocytes in a functional and viable state.

Studies on the metabolism and excretion of Benzo(a)pyrene in isolated adult rat hepatocytes

Biphenyl metabolism in isolated rat hepatocytes: Effect of induction and nature of the conjugates

1. The metabolism of biphenyl by isolated viable rat hepatocytes has been studied and a tentative scheme of metabolism proposed which involves initial hydroxylation at the 2- and 4-positions followed by conjugation and /or further hydroxylation of these primary metabolites.2. Biphenyl was toxic to viable hepatocytes when used at a concentration approaching that used in conventional microsomal assay systems.3. The production of small amounts of 4-hydroxybiphenyl appears to activate its subsequent conjugation.4. The data presented in this paper integrate previous results obtained with cell fractions, and demonstrates the importance of the isolated, viable hepatocyte system as a model for total drug metabolism.

The Metabolism of Biphenyl by Isolated Viable Rat Hepatocytes

1. The metabolism of a number of xenobiotics has been investigated in isolated viable rat hepatocytes and kidney tubule fragments in suspension.2. The level of Phase I metabolism was very low in the kidney cells although such cells possess appreciable Phase II metabolic capacity.3. These findings are discussed in relation to the physiological role of cytochrome P-450 and the conjugating enzymes, and the value of intact cell systems in assessing inter-organ differences in xenobiotic metabolism.

Jeffrey R. Fry

Papers

The enzymic isolation of adult rat hepatocytes in a functional and viable state.

Studies on the metabolism and excretion of Benzo(a)pyrene in isolated adult rat hepatocytes

Biphenyl metabolism in isolated rat hepatocytes: Effect of induction and nature of the conjugates

The Metabolism of Biphenyl by Isolated Viable Rat Hepatocytes

A comparison of drug-metabolizing capability in isolated viable rat hepatocytes and renal tubule fragments.