Preparation of Parenchymal and Non-Parenchymal Cells from Adult Human Liver — Morphological and Biochemical Characteristics

Abstract

Summary: By perfusion of the isolated human liver with collagenase and hyaluronidase a mixed suspension of various cell types was obtained. Pure parenchymal cells were prepared by differential centrifugation, pure non-parenchymal cells by the use of pronase and subsequent isopycnic centrifugation on metrizamide gradients (50-300 g/1). About 90% of the parenchymal and non-parenchymal cells were viable as judged by tiypan blue staining. Non-parenchymal cells were not capable of gluconeogenesis but utilized glucose at high rates. Parenchymal cells retained their ability to form glucose and to accumulate glycogen from fructose > lactate/pyruvate > alanine. Studies on binding of 125 Ilabelled insulin by isolated parenchymal cells were performed at 30 °C. The binding data may fit a model with a minimum of two classes of binding sites: (a) high affinity -low capacity sites (Kd ~~ 6.6 nmol/1, capacity ~ 16000 insulin molecules per cell) and (b) low affinity -high capacity sites (K d ~ 0.37 / , capacity ~ 646 000 molecules per cell).

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

“…The isolation of primary human hepatocytes was performed for the first time more than 40 years ago (Bojar et al 1976; Strom et al 1982; Guguen-Guillouzo et al 1982; Reese and Byard 1981). The introduction of the two-step isolation procedure using collagenase by Seglen (1976) signified an important progress in primary liver cell isolation.…”

Drug metabolising activity of freshly isolated human hepatocytes.

“…The older the cells, the more substantial was this loss in viability. In contrast to the hepatocytes immediately after isolation, there was a tendency for older cells to maintain (Bojar et al, 1976;Miyazaki et al, 1981;Strom et al, 1982). The most successful methods appear to be those using a modification of the collagenase perfusion technique of Berry & Friend (1969 (Miyazaki et al, 1981;Maekubo et al, 1982;Strom et al, 1982;Guguen-Guillouzo et al, 1982).…”

“…For these reasons it is desirable to be able to study metabolism and toxicity in human isolated hepatocytes. Several groups have investigated methods for the isolation of human hepatocytes (Bojar et al, 1976;Miyazaki et al, 1981;Strom et al, 1982) but there have been very few reports on their drug metabolising activity. Bojar et al (1976) perfused the whole liver of a renal transplant donor and obtained cells of high viability.…”

Cell Transplantation: A Possible Alternative to Orthotopic Liver Transplant (OLT)

“…In 1976, the traditional two-step collagenase perfusion technique was developed for rat tissue (Seglan, 1976), which was later adapted for use with human tissue (Bojar et al, 1976). Another widely used method, which yields a high number of viable cells per gram of whole liver tissue, is the three-step collagenase perfusion technique (Dorko et al, 1994;Nakazawa et al, 2002;Mitry et al, 2004;Alexandrova et al;.…”