Håkan Pertoft

Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence.

A rapid method for mass isolation of functionally intact hepatocytes and reticuloendothelial cells from a single rat liver is described. The technique is based on collagenase perfusion of the liver, isopycnic sedimentation in Percoll, and selective adherence of the cells. The Kupffer cells (KC) attach and spread on glass or plastic in serum‐free medium 15 min following seeding. Cultures of KC are 90%–95% pure with about 5% liver endothelial cells (LEC), <1% parenchymal cells (PC) and a maximum of 5% stellate cells (SC). The LEC adhere and spread on fibronectin 60–120 min following seeding, forming cultures that are contaminated with 5–10% SC and <1% KC and PC. The yield of plated LEC is 50–60 × 106 per 200‐g rat. Ultrastructural analysis shows that Percoll does not associate with the cells during the separation procedure.

Endothelial cells are a site of uptake and degradation of hyaluronic acid in the liver

Abstract In a recent communication it was shown that intravenously injected radioactively labelled hyaluronic acid was preferentially taken up by the liver and degraded. We now report that uptake occurs in the liver endothelial cells and that these cells degrade the polysaccharide in vitro into low-molecular weight (LMW) products.

Density gradients prepared from colloidal silica particles coated by polyvinylpyrrolidone (Percoll)

Abstract A new gradient medium (Percoll) for density gradient centrifugation of cells and subcellular particles is described. It consists of colloidal silica particles which have been firmly coated with a layer of polyvinylpyrrolidone. The particle population is polydisperse, and the average diameter is approximately 17 nm. Both the electrophoretic mobility of the particles and the conductivity of the solution are low, indicating a low surface charge. The colloid has a high solubility and forms clear solutions. It can be used in concentrations which give solution densities between 1.00 and 1.20 g/ml. The solutions have a low osmolality and can be mixed with electrolytes of physiological pH and ionic strength. The sedimentation properties of the particles are described. Density gradients can be formed by highspeed centrifugation. The colloid has been shown to be nontoxic for a number of cells and cell organelles.

The viability of cells grown or centrifuged in a new density gradient medium, Percoll(TM).

A new density gradient medium, Percoll (a modified colloidal silica), has been tested for toxicity in primary cultures of rat liver and calf testicle cells, and in continuous cultures of pig kidney and HeLa cells. The presence of Percoll did not appreciably affect the growth or viability of the cells as judged from cell counts and morphology. The various cells were also centrifugea in gradients of Percoll and subsequently cultured. The in vitro growth of the cells was similar to that of untreated cells. Rat liver cells were labelled in vivo with [125I]asialoceruloplasmin (parenchymal cells) or heat-denatured [125I]albumin (non-parenchymal cells). After dispersion of the cells and iso-pycnic centrifugation in Percoll the non-parenchymal cells banded preferentially at a lower density (1.04−1.05 g/ml) than parenchymal cells (1.07−1.09 g/ml). The two types of cells showed very different morphology in cell culture. The non-parenchymal cells retained their phagocytic properties during culture. Injured cells and cell debris band at the top of the Percoll gradients in contrast to their behaviour in gradients containing low molecular weight substances. Centrifugation in Percoll can be used to enrich viable cells.

Fractionation of cells and subcellular particles with Percoll.

At present, centrifugation is the most common method for separation and isolation of cells and subcellular particles. The technique can be used for a wide range of applications. During latter years it has become obvious what a powerful method density gradient centrifugation is, especially when used in conjunction with sensitive assays or clinical treatments. The most active areas for use of density gradient centrifugation include purification for in vitro fertilization of sperm of both human and bovine origin, isolation of cells for cell therapy of patients receiving chemo- and radiation therapy and basic research both on cellular and subcellular levels. These treatments and investigations require homogeneous populations of cells and cell organelles, which are undamaged after the separation procedure. Percoll, once introduced to reduce convection during centrifugation, has proved to be the density gradient medium of choice since it fulfills almost all criteria of an ideal density gradient medium. Recently good results have also been obtained after silanization of colloidal silica particles, e.g. BactXtractor. The latter medium has proved to be useful in recovery of microorganisms from food samples free of inhibitors to the Polymer Chain Reaction (PCR). The separation procedures described for Percoll in this review seem to be applicable to any cells or organelles in suspension for which differences in size or bouyant density exist. Furthermore, since Percoll media are inert, they are well suited for the separation of fragile elements like enveloped viruses.

Functional and morphological characterization of cultures of Kupffer cells and liver endothelial cells prepared by means of density separation in Percoll, and selective substrate adherence

SummaryThis paper presents a study on the structure and function of Kupffer cells (KC) and liver endothelial cells (LEC) isolated by a simple and rapid technique involving 1) perfusion of the liver with collagenase; 2) cell separation by means of density centrifugation in Percoll; and 3) cell culture, taking advantage of the fact that KC and LEC differ in their preferences for growth substrate. The KC, which attach and spread under serum-free conditions on surfaces of glass or plastic during the first 15 min in culture exhibit a typical macrophage-like morphology including membrane ruffling and a heterogenous content of vacuoles. Moreover, these cells express (a) Fc receptors (FcR) for binding and phagocytosis of erythrocytes covered with immune globulin G (E-IgG), and (b) complement receptors (CR) for binding and serum dependent phagocytosis of erythrocytes covered with either human C3b or mouse inactivated C3b (iC3b). The cells also bind fluid phase fluoresceinated C3b. Approximately 30% of the KC express immune response-associated (Ia)-antigens.The LEC attach and spread on fibronectin coated surfaces, but not on glass or plastic surfaces, during the first two hours in culture with or without serum, and are morphologically distinct from KC. Cultured LEC are well spread out with no membrane ruffling and with numerous large vesicles surrounding the regularly shaped nucleus. These cells bind, but do not ingest E-IgG via the FcR, but no binding of fluid phase C3b or particle fixed C3b or iC3b can be observed. Incubation of LEC with fluorescein amine conjugates of ovalbumin or formaldehyde treated serum albumin, but not with fluoresceinated native serum albumin, results in accumulation of fluorescence specifically localized in the large perinuclear vesicles. Neither KC nor any other cell types tested have the ability to accumulate fluorescence upon incubation with these compounds. Iaantigens are not present on the LEC.Cytochemical demonstration of unspecific esterase, acid phosphatase, and peroxidase reveals different patterns and intensities of staining in KC as compared to LEC.

Separation of various blood cells in colloidal silica-polyvinylpyrrolidone gradients.

Abstract New methods are described for the separation of the cellular components of human blood. They can be separated into erythrocytes, polymorpho-nuclear leucocytes, mononuclear leucocytes and platelets by centrifugation in density gradients of colloidal silica and polyvinylpyrrolidone. The procedures are simple and the yields and resolution achieved higher than in methods currently available. The fractionations are made in media of physiological pH and osmolality. The cells are intact after the fractionation as measured by the fluorescein diacetate accumulation test, the trypan blue exclusion test and thymidine incorporation in the lymphocyte fraction after phyto-hemagglutinin stimulation. In the techniques described, between 1 and 200 ml of blood can be fractionated at a time. Alternatively, the different cell populations may be banded at the interface between silica solutions of different densities (on density cushions). The latter technique is especially suitable for working up larger quantities of blood.

Separation and Characterization of Liver Cells

Publisher Summary This chapter discusses the separation and characterization of liver cells. The methods of preparation of isolated liver cells involve treatment with enzymes, separation by centrifugation, and selective substrate attachment. The other developed method involving collagenase perfusion, isopycnic centrifugation in Percoll, and selective substrate attachment is rapid and gives high-yield and high-purity preparations of functionally intact cells. A rapid and frequently used method for assessing cell viability is based on the capacity of cells to exclude trypan blue. However, this method gives an indication only of the integrity of the plasma membrane. Cells that exclude trypan blue may prove to be nonviable when tested for functional or morphological properties. Light microscopy forms the basis of most methods of identification of liver cells. Electronic cell counters are certainly useful, but only visual inspection with the aid of a hemocytometer and a phase-contrast microscope give information about the presence of aggregates, cell debris, and PC-derived particles. Cytochemical staining for endogenous peroxidase is the most frequently used means to positively distinguish KC from LEC and SC in the rat.

Isopycnic Separation of Cells and Cell Organelles by Centrifugation in Modified Colloidal Silica Gradients

Methods that under mild conditions can be used to purify cells and cell organelles have played a major role in the progress of cell biology and molecular biology. Since the tissues of higher organisms usually are made up of more than one cell type there is a need for methods that can isolate particular cells from mixtures. Similarly, cell homogenates must be fractionated before specific cell organelles can be studied.

Differential expression of platelet-derived growth factor α- and β-receptors on fat-storing cells and endothelial cells of rat liver☆

Abstract Fat-storing cells and endothelial cells of the liver sinusoids play important roles in the biosynthesis and degradation of hyaluronan (HYA). These cells were isolated from rat liver by a simple and rapid procedure involving: (1) cell separation by centrifugation on a Nycodenz gradient, after dispersion of the liver cells by collagenase perfusion; (2) further purification of the cells by centrifugation on a discontinuous Percoll gradient; and (3) culturing of the cells, taking advantage of the different time that seeded cells need for attachment to plastic surfaces. We have determined the effects of two isoforms of platelet-derived growth factor (PDGF), PDGF-BB and PDGF-AA, on HYA production by the original fat-storing cells, as well as by fat-storing cells which had changed in vitro to myofibroblast-like cells. PDGF-BB was found to stimulate HYA synthesis in both types of cells with a maximal response equal to that obtained with 10% fetal calf serum. PDGF-AA had no stimulatory effect on HYA production. Fat-storing cells and their modified myofibroblast-like phenotype bound specifically to 125I-PDGF-BB, but not to 125I-PDGF-AA, indicating that they had PDGF β-receptors, but not α-receptors. In contrast, liver endothelial cells were found to have PDGF α-receptors, but not β-receptors.