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.

Localization and quantitation of hyaluronan and sulfated glycosaminoglycans in the tissues and intraluminal fluid of the pig oviduct.

Glycosaminoglycans (GAGs), hyaluronan (HA) and heparan sulfate (HS) were localized in the pre- and post-ovulatory oviducts of inseminated and control (non-inseminated) sows using biotinylated HA-binding protein (HABP) and anti-syndecan antibodies respectively. In addition, the concentrations of HA and total sulfated GAGs (S-GAGs) were measured in fluid collected in vivo from either a selected tubal segment (isthmus or ampulla) or from the contralateral whole oviduct (WO) of non-inseminated sows during proestrus-metoestrus. HA was localized in the lamina propria of the entire oviduct, but epithelial HA-labelling was only present in the sperm reservoir (uterotubal junction adjacent isthmus) in control and inseminated sows. In contrast, immunolabelling for HS proteoglycans (HSPGs, syndecans) was present on the entire epithelial lining, both pre and post ovulation and in both sow groups. Both HA and S-GAGs could be detected in the intraluminal fluid. Concentrations varied among sows and segments; those of the S-GAGs being higher (P<0.05) than that of HA. Mean levels of S-GAGs and HA tended to increase in the fluid collected from isthmus and ampulla during standing oestrus. Fluid levels from the WO, however, fluctuated less during the collection period. Major statistical differences were not present, owing to the large variation seen between animals. The results confirm, however, that GAGs are present in the pig oviduct. The conspicuous localization in the sperm reservoir and the tendency to higher levels in the fluid during pre-ovulatory oestrus support the hypothesis that GAGs play a role in modulating sperm viability and capacitation during sperm transport in the pig oviduct.

Evaluation of sperm damage and techniques for sperm clean-up.

The cryopreservation of bull semen adversely affects the metabolism, motility and membrane integrity of the spermatozoa, thereby decreasing the fertilizing ability of the processed sample. The present review covers methods available for examining the functionality of bull spermatozoa after thawing, including the use of fluorophores in combination with morphological examination. Procedures for clean-up, separation and selection of morphologically-normal, viable spermatozoa in vitro are also reviewed. Among the reviewed procedures are methods for dilution and washing and the selective fractionation of sperm subpopulations (especially density-gradient centrifugation in silica-bead suspensions), as well as methods based on adherence to glass (differential filtration) and sperm self-migration (particularly swim-up through hyaluronic acid). Emphasis is placed on assessing the value of these techniques for diagnostic purposes and for optimizing the methodology of in vitro fertilization.

Hyaluronan oligosaccharides induce tube formation of a brain endothelial cell line in vitro.

In remodeling tissues the formation of new blood vessels is an essential process which is regulated by different factors. During such processes an increase in hyaluronan synthesis and turnover is often seen and recent observations have suggested that hyaluronan can both promote and inhibit neovascularization depending on its molecular mass. In this work we show that a brain capillary endothelial cell line forms tubes in a collagen gel after stimulation by hyaluronan oligosaccharides. Ultrastructural examination of the tubes by transmission electron microscopy revealed that the cord-like outgrowths consisted of 2-10 tightly packed cells containing a continuous lumen. The tube formation in response to hyaluronan oligosaccharide was not mediated by activation of receptors for fibroblast growth factor. The endothelial cell line, which does not synthesize hyaluronan, exhibited specific hyaluronan-binding sites, with about 3000 hyaluronan molecules (M(r) 3.85 x 10(6)) bound per cell at saturation and Kd was 0.05 x 10(-9) M. Furthermore, the cell line was stained with mAb IVd4 that recognizes a novel class of hyaluronan-binding proteins and mAb IM7 which recognizes CD44 molecules.

Recruitment of type I collagen producing cells from the microvasculature in vitro.

We have previously suggested that microvascular pericytes can differentiate into fibroblast-like, type I collagen-producing cells during excessive dermal scarring in vivo (Sundberg, C., Ivarsson, M., Gerdin, B., and Rubin, K., Lab. Invest. 74, 454-468, 1996). Here we have investigated to what extent pericytes derived from microvessels of full-term human placenta exhibited this capacity in vitro. Vascular fragments of human term placenta were isolated by enzymatic digestion and separation in Percoll. Their microvascular origin was ascertained by confocal microscopy using antibodies specific for endothelial cells (PAL-E) and pericytes (high-molecular-weight-melanoma-associated antigen). When vascular fragments were cultured in vitro, large cells with irregular edges migrated out from the fragments. After 4-6 days in culture, these cells started to proliferate and reached near confluence after approximately 8 days. The cultures were not overgrown by clones of cells with a high proliferative capacity, as demonstrated by cell membrane fluorescence staining and Ki67 expression. Expression of PAL-E, high-molecular-weight-melanoma-associated antigen, smooth muscle alpha-actin, desmin, and collagen synthesis (prolyl-4-hydroxylase and type I procollagen, as well as collagen pro-alpha1(I) mRNA) were followed during a culture period of 8 days. The cells were PAL-E negative but expressed high-molecular-weight-melanoma-associated antigen, smooth muscle alpha-actin, and desmin. Based on morphology and expression of the various markers, the outgrowing cells were identified as pericytes. With time in culture the cells decreased their expression of all these markers and increased their expression of prolyl-4-hydroxylase, type I procollagen, and collagen pro-alpha1(I) mRNA. Metabolic labeling and SDS-PAGE analysis of labeled proteins revealed that type I collagen was the major collagen species synthesized in the cultures. Our results support the hypotheses that pericytes can leave the vasculature and differentiate into collagen-producing cells and that cultured "fibroblasts" are derived from pericytes.

Biosynthesis and degradation of hyaluronan by nonparenchymal liver cells during liver regeneration.

Hepatic stellate cells (HSC) and endothelial cells of the liver sinusoids synthesize and degrade hyaluronan, respectively. The roles of these cell types in the biosynthesis and degradation of hyaluronan were studied during regeneration following partial hepatectomy. Pure cultures of HSC and liver endothelial cells (LEC) were obtained from regenerating liver at different stages using a Nycodenz gradient followed by discontinuous Percoll gradient. The HSC that established 3 or 4 days after partial hepatectomy synthesized large amounts of hyaluronan when cultured in the presence of fetal calf serum (FCS) or platelet-derived growth factor B-chain homodimer (PDGF)-BB. These cells, as well as LEC, expressed active PDGF beta-receptors. Furthermore, the ability of LEC to degrade hyaluronan was decreased at early stages of liver regeneration. The increased synthesis of hyaluronan by HSC and the failure of LEC to catabolize the polysaccharide resulted in elevated hyaluronan concentrations in the blood.

Chondroitin sulfate proteoglycan modulates the permeability of hyaluronan-containing coats around normal human mesothelial cells.

The composition and permeability of the pericellular coat surrounding normal human mesothelial (NHM) cells have been studied in vitro. NHM cells were grown in the presence of 3H-glucosamine and the amount of label recovered in hyaluronan and chondroitin sulfate was determined after selective enzymatic digestion of the polysaccharides in medium, pericellular, and intracellular pools. For comparison a similar analysis was carried out on mesothelioma cells (Mero-14). Of the labeled polysaccharides in the medium and pericellular pools of NHM cells about 80-90% could be ascribed to hyaluronan and only 3-5% to chondroitin sulfate. In contrast, Mero-14 synthesized only minute amounts of hyaluronan whereas chondroitin sulfate corresponded to 61% of the total glycosaminoglycans in the culture. The results exclude a structure of the pericellular layer of NHM cells similar to the hyaluronan-proteoglycan aggregates found in cartilage. The permeability of the pericellular layer was tested by the exclusion of polystyrene microspheres and bacteria of diameter 0.1-3.0 microns, as well as erythrocytes of diameter 7 microns. While the erythrocytes were excluded the smaller particles penetrated the coat. By adding 0.5 mg/ml of aggregating cartilage proteoglycan to the medium particles of 0.3 microns or larger were also excluded. Thus exogenous proteoglycans can reinforce the structure of the pericellular layer.

Inter-alpha-inhibitor is required for the formation of the hyaluronan-containing coat on fibroblasts and mesothelial cells.

Cultured cells of various origins have been shown to be surrounded by a hyaluronan-containing coat, a structure that can be visualized by its ability to exclude large particles such as erythrocytes. When cultured in medium with no or low concentrations of serum, the cells lose their coats, although they still produce hyaluronan; upon the addition of serum, the coats are formed again. Here, we show that the serum protein inter-alpha-inhibitor can replace whole serum as an inducer of the formation of the coats on fibroblasts and mesothelial cells. The physiological role of inter-alpha-inhibitor has so far been unclear; our findings, together with those obtained with cumulus cell-oocyte complexes (Chen, L., Mao, S.J., and Larsen, W. J. (1992) J. Biol. Chem. 267, 12380-12386), suggest that inter-alpha-inhibitor and related proteins have a general function as stabilizers of hyaluronan-containing pericellular coats.

Synthesis and assembly of the hyaluronan-containing coats around normal human mesothelial cells.

In this study we examined the capacity of normal human mesothelial (NHM) cells and human malignant mesothelioma cells to form hyaluronan-containing pericellular matrices or "coats." The assembly of the pericellular coats was visualized by a particle exclusion assay. We found that large hyaluronan-containing coats were formed around NHM cells whereas their transformed counterparts had no or very limited coats. The coats were removed by treatment with Streptomyces hyaluronidase, which specifically degrades hyaluronan. NHM cells exhibited hyaluronan-containing pericellular matrix within 5 h after seeding. The formation of the coats was stimulated by platelet-derived growth factor and epidermal growth factor. Interestingly, the assembly of the hyaluronan-dependent pericellular matrices was inhibited by the addition of hyaluronan dodecasaccharides. The inhibitory effect on the formation of the coats was due to a destabilization of pericellular matrix and not due to an inhibitory effect of hyaluronan dodecasaccharides on hyaluronan synthesis. In contrast, hyaluronan hexasaccharides, an inhibitor of the interaction between polymeric hyaluronan and its cell surface receptors, had no effect on the size of the coat. Thus, our results are compatible with the possibility that the pericellular matrix surrounding NHM cells consists of newly synthesized hyaluronan which is extruded from the cell and independent of hyaluronan receptors on the cell surface. The coat seems to be stabilized by interactions (hyaluronan-hyaluronan or hyaluronan-protein bridges) which can be prevented by hyaluronan dodecasaccharides.

Hyaluronan synthesis by rat liver stellate cells is enhanced under endotoxic conditions.

As part of an investigation of the effect of sepsis on the sinusoidal cells of liver we studied the influence of conditioned media from Kupffer cells (KC) and liver endothelial cells (LEC) from normal rats and animals pretreated with endotoxin (ET) on the hyaluronan (HA) production by stellate cells (SC) in culture. SC proliferation, as measured by direct cell counting and [3H]thymidine incorporation, was also recorded. An SC from an ET-treated rat produces similar amounts of HA as a normal SC when grown in culture medium containing 10% fetal calf serum (FCS). When animals were treated with ET, there was, however, an increase in the yield of isolated SC and thus the whole cell population in ET-treated rats has a potential to produce a larger amount of the polysaccharide. Factors present particularly in the KC conditioned media supplemented with 10% FCS from normal and endotoxemic animals greatly stimulated the proliferation and synthesis of HA in SC cultures, after a lag-phase which was four days for normal SC and three days from ET-treated animals. The rate of HA synthesis was closely related to the increase in the amount of thymidine incorporation into DNA. Conditioned media containing 10% FCS obtained from LEC cultures were also stimulatory but were less effective in inducing cell proliferation and production of HA. We suggest that the elevation of plasma HA found in severe endotoxemia and sepsis may be caused in part by the: (i) increased number of SC; (ii) an in vivo activation of SC population which predisposes for HA-production; and, (iii) action of factors or mediators from cells of the reticuloendothelial system of the liver on the cell proliferation and on the HA synthesis in the SC population.

Endocytosis of hyaluronan in rat Kupffer cells.

The binding, uptake and degradation of hyaluronan (HA) labelled with 3H in its acetyl group were studied in cultured rat Kupffer cells (KC). At 4 degrees C the binding increased with increasing concentrations of HA in the culture medium up to at least 1 microgram/ml, when saturation occurred. Binding could be prevented efficiently by the addition of an excess of unlabelled HA, and to a lesser extent by chondroitin sulphate and oligosaccharide fragments of HA, consisting of four sugars or more. The labelled HA bound to the cells could be removed by incubating the cells with Streptomyces hyaluronidase, or trypsin, indicating that the HA-binding sites are located on the cell surface. At 37 degrees C HA was internalized in a concentration-dependent manner, and degradation products appeared in the supernatant after 1-5 h, depending on the concentration applied. At 50 ng of free HA/ml, each KC accumulated 60 ag of the polysaccharide/min in the first 1 h, and degraded a total amount of 10 fg of HA during an 8 h period. Addition of the negatively charged polysaccharide dextran sulphate reduced binding, and to an even greater extent internalization, of HA in KC, while no effect was observed with dextran. Depletion of intracellular potassium caused a marked reduction in the rate of endocytosis of cell-membrane-associated HA into KC, without affecting binding. Addition of KCl to the culture medium returned endocytosis of [3H]HA to normal levels. There was no effect on binding and a partial effect on internalization by depletion of bivalent cations or in the presence of EDTA. The degradation of [3H]HA by KC cultures was abolished in the presence of weak bases, NH4Cl and chloroquine, supporting the idea that HA is endocytosed into lysosomes prior to degradation. The fluid-phase marker [14C]sucrose was internalized in the cells at much lower rate than was HA. Rates of binding, internalization and degradation of HA in KC point therefore to a specific endocytosis followed by an intracellular degradation to low-M(r) compounds. It was estimated that, under physiological conditions, KC only clear a minor proportion of circulating HA.

Uptake of hyaluronan in hepatic endothelial cells is not directly affected by endotoxin and associated cytokines.

The uptake of hyaluronan (HYA) labeled with 3H in its acetyl group was measured in cultured liver endothelial cells from normal rats and from rats previously treated with sublethal doses of Escherichia coli endotoxin (ET). Replicate cultures were also exposed to recombinant human tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) or interferon-gamma for 1 to 3 h before the measurement of hyaluronan uptake. Under all conditions, HYA was absorbed by endothelial cells at rates consistent with receptor-mediated absorption. In cells exposed to HYA 20 h after isolation, rate of uptake was less than half the rate in cells exposed 6 or 7 h after isolation. Cellular uptake of HYA was neither reduced nor enhanced by any of the treatments with cytokines. Prior exposure of the cell donors to ET caused a three-fold increase in their plasma HYA but did not alter the subsequent rate of cellular HYA uptake in vitro, either with or without added treatment with TNF-alpha or IL-1. It was concluded that the elevation of plasma HYA caused by septicaemia or by the experimental administration of ET or TNF-alpha cannot be attributed to direct interference with HYA receptors on hepatic endothelial cells.

Further characterization of carboxymethylated aspartic acid agarose. Purification of human alpha 2-macroglobulin and hemopexin.

alpha 2-Macroglobulin and hemopexin were purified by affinity chromatography on a recently introduced chelating matrix, i.e., carboxymethylated aspartic acid agarose, coupled with cobalt(II). Adsorption was performed at neutral pH and the proteins were eluted by lowering the pH to 5.0. An alternative method for desorption as well as comparison with iminodiacetic acid agarose coupled with cobalt(II) is also described.

Differential expression of platelet-derived growth factor alpha- and beta- receptors on fat-storing cells and endothelial cells of rat liver.

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 beta-receptors, but not alpha-receptors. In contrast, liver endothelial cells were found to have PDGF alpha-receptors, but not beta-receptors.

Purification of factor VIII:c coagulant activity from rat liver nonparenchymal cell culture medium by immobilized metal ion affinity chromatography.

The purification of factor VIII:c coagulant activity on the basis of its affinity for calcium is described. For this purpose, use was made of a recently introduced chelating matrix, i.e., carboxymethylated aspartic acid agarose, coupled with calcium--thereby creating a gel with specificity comparable with biospecific affinity chromatography. In a single step factor VIII:c activity was purified from rat liver nonparenchymal cell culture medium with a purification factor of 85-fold. The material exhibits a single band on polyacrylamide gel electrophoresis.

Isolation of the perisinusoidal vitamin A-storing cells from rat liver on Ca2(+)-immobilized glass surfaces.

Calcium was immobilized on glass slides employing covalently coupled iminodiacetic acid. The calcium-coated glass surfaces were then used for purification and culturing of perisinusoidal vitamin A-storing cells from rat liver. The cells were isolated in a yield of 0.9 x 10(6) cells/g rat liver without cross-contamination by other hepatic cell types. The cells were characterized by morphology, vitamin A fluorescence, and immunohistochemistry to detect expression of cellular retinol-binding protein.

N-acetylglucosamine-6-phosphate deacetylase in hepatocytes, Kupffer cells and sinusoidal endothelial cells from rat liver.

The activity of N-acetylglucosamine-6-phosphate deacetylase, a key enzyme in the pathway of N-acetylglucosamine catabolism, was measured in hepatocytes, Kupffer cells and sinusoidal endothelial cells from rat liver and cultured human skin fibroblasts. Kupffer cells and endothelial cells had similar high levels of deacetylase activity that were more than twice the level observed in fibroblasts. In contrast, hepatocytes had extremely low activity (several hundredfold less than Kupffer cells and endothelial cells). A major implication of deacetylase deficiency in hepatocytes is that N-acetylglucosamine generated as a result of the catabolism of complex carbohydrates in these cells cannot enter glycolysis and must be largely reused for the synthesis of plasma glycoproteins and other N-acetylglucosamine-containing macromolecules.

Purification of human serum amyloid P component (SAP) by calcium affinity chromatography.

Serum amyloid P component (SAP) has been purified from human serum by means of immobilized metal ion affinity chromatography (IMAC). It was selectively concentrated on carboxymethylated aspartic acid agarose (CM-Asp-agarose) loaded with calcium and, employing very mild conditions, purified to electrophoretical and immunological homogeneity in a single step amounting to about 1900-fold purification. As a purification method our procedure thus compares well with bio-specific affinity chromatography.

Enzymic pathways of hyaluronan catabolism.

The enzymic degradation of hyaluronan in mammalian tissues takes place in two phases, encompassing breakdown of the polysaccharide to its monosaccharide constituents and subsequent utilization of the monosaccharide products. Degradation to the monosaccharide components is effected by the concerted action of three enzymes, hyaluronidase, beta-D-glucuronidase and beta-N-acetyl-D-hexosaminidase. The relative contributions of hyaluronidase and the two exoglycosidases to the physiological catabolism of hyaluronan are not yet known but consideration of the kinetic properties of the three enzymes clearly indicates that hyaluronidase is best suited for the initial attack on the polysaccharide, inasmuch as its Km for hyaluronan is 1000- to 10,000-fold lower than that estimated for beta-D-glucuronidase. Recent investigations in the authors' laboratories have been focused on the catabolism of hyaluronan and other complex carbohydrates in liver, since the sinusoidal endothelial cells in this organ are the main sites for degradation of circulating hyaluronan. Assay of ten lysosomal hydrolases in isolated rat liver cells showed considerably higher activities in Kupffer cells and endothelial cells than in hepatocytes for nine of the enzymes, including beta-D-glucuronidase and beta-N-acetyl-D-hexosaminidase. The activity of N-acetylglucosamine-6-phosphate deacetylase, a key enzyme in the metabolism of the N-acetylglucosamine released by the lysosomal degradation of hyaluronan and other complex carbohydrates, has also been determined. High deacetylase activities were observed in both Kupffer cells and endothelial cells but, surprisingly, virtually no activity was detected in hepatocytes. This finding implies that N-acetylglucosamine cannot be degraded in hepatocytes and must be largely reutilized in the synthesis of new macromolecules. Further studies of the enzymes involved in hyaluronan degradation and N-acetylglucosamine utilization in the liver are under way.