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Cell Biology International (2003) 27, 67–73 (Printed in Great Britain)
Isolation of rat Kupffer cells: a combined methodology for highly purified primary cultures
V. Valatas, C. Xidakis, H. Roumpaki, G. Kolios* and E.A. Kouroumalis
Liver Research Laboratory, Faculty of Medicine, University of Crete, P.O. Box 1393, Heraklion, GR-71409 Crete, Greece


Abstract

We report a four-step procedure that optimizes the methodology for isolation of highly purified rat Kupffer cells (KC). We combined the previously reported techniques of enzymatic tissue treatment, density gradient centrifugation, centrifugal elutriation and selective adherence. ED-2 immunophenotyping and non-specific esterase histochemistry were used for cell identification. This combination resulted in a satisfactorily high yield of 80–100×106KCs per liver, over 95% positive for ED-2 and 98% viable cells. Cultures of isolated KCs were functionally intact and exhibited a concentration and time-dependent LPS-induced TNF-α and nitric oxide production.


Keywords: Kupffer cells, Sinusoidal liver cells, Cell isolation, Cell culture.

*Corresponding author. Tel./fax: +30-2-81-054-2085


1 Introduction

Liver sinusoidal lining contains the main non-parenchymal cells of the liver, that is the Kupffer cells (KC), the sinusoidal endothelial cells (SEC) and the stellate cells (SC), each representing about 30, 60 and 10% of the non-parenchymal cell population, respectively. All three cell-types play a crucial role in liver homeostasis as well as in initiation, maintenance and outcome of liver inflammation (Smedsrod et al., 1994). In vitro studies using primary cultures are a valuable tool for the exploration of specific immunological functions and the clarification of distinct roles of these cells.

Various methods have been previously described for the isolation and purification of the different sinusoidal cells of the liver. However, single step procedures, like density gradient centrifugation or centrifugal elutriation, alone are unable to eliminate the contamination of KC cultures from other non-parenchymal cell types owing to the fact that the densities as well as the cell size of the sinusoidal cells show a significant overlap (Bøyum et al., 1983; Yata et al., 1999). Based on a combination and appropriate modification of previously reported techniques (Blomhoff et al., 1984; Knook et al., 1977; Zahlten et al., 1978), we report in detail a reliable and reproducible methodology for the isolation of KC, which gives highly purified and functionally intact cell cultures.

2 Methods

2.1 Sinusoidal cell isolation

KC were isolated from pathogen-free male Sprague–Dawley rats over 12 months old (450–600g). A modification of the combined ‘collagenase–pronase’ perfusion method was used to dissociate liver tissue (Zahlten et al., 1978). In detail, animals were anaesthetized by an intraperitoneal injection of Pentobarbital (50mg/kg), the portal vein was cannulated with a 22 G catheter and the liver was perfused in situ with 200ml Hanks' balanced salt solution (HBSS) Ca2+/Mg2+-free (GibcoBRL,Paisley, UK) at 10ml/min, 37°C in a non-recirculating fashion. The liver was then excised, transferred to a 35mm culture dish and perfusion was continued ex situ with 60ml 0.2% pronase (Boehringer–Mannheim, Mannheim, Germany) in HBSS (GibcoBRL) followed by 225ml 0.01% collagenase (Boehringer–Mannheim) in HBSS under the similar conditions.

The organ was then detached from the perfusion device, the capsule was removed and the tissue minced to small pieces. Tissue was dispersed in 100ml HBSS containing 0.03% pronase and 0.01% DNAse (Boehringer–Mannheim) and was incubated under constant agitation at 37°C for 30min. Following digestion, the liver homogenate was filtered through an 125μm nylon mesh to remove undigested tissue and the cell suspension was centrifuged at 400×g for 7min at 4°C.

Cell pellet was subsequently resuspended in HBSS and mixed with 29.4% Iodixanol working solution (Optiprep™, Nycomed-Pharma, Oslo, Norway) to give an 11.7% (d=1.07gr/ml) gradient. This was carefully layered onto a 17.6% (d=1.097gr/ml) Iodixanol gradient prepared by mixing 12ml of Iodixanol working solution with 8ml HBSS. The double layer discontinuous gradient formed was carefully overlaid with 0.5ml of HBSS and centrifuged at 1400×g for 17min at 4°C without applying the centrifuge brake. After centrifugation, two layers were formed; one at the top of the 11.7% gradient mostly containing non-parenchymal cells and a second at the interface between the two gradients mostly containing viable hepatocytes and non-parenchymal cells of higher density. The remaining pellet contained the majority of the enzymatically injured hepatocytes. Interface and top layer were both collected, resuspended in HBSS and centrifuged at 400×g for 7min at 4°C. The sinusoidal-cell enriched pellet was resuspended in 20ml of HBSS containing 0.05% DNAse and collected with a syringe through a 20 G needle to disperse cell clumps.

2.2 KC purification

2.2.1 Centrifugal elutriation

KC were further separated from viable hepatocytes and other sinusoidal cells by a modification of the centrifugal elutriation method originally described byKnook et al. (1977). The elutriation system consisted of a J2-MC centrifuge (Beckman, Paolo Alto, California, USA) with a JE-6B rotor equipped with a standard chamber (Beckman) and linked to a high precision pump (Masterflex 7521-25, Cole Parmer, Chicago, USA). The elutriation was performed at a rotor speed of 2500rpm at 25°C. HBSS was used as elutriation medium. Aliquots (10ml) of the cell suspension were introduced into the elutriation system at a flow rate of 18.5ml/min. After 200ml of medium had passed through and a cell pellet was clearly formed into the elutriation chamber, the flow rate was increased stepwise from 18.5 to 25, 35, 45, 60, 80 and 100ml/min. A volume of 100ml of cell suspension was collected at each point.

2.2.2 Adherence to plastic

To purify the obtained cell population further, we used the method of selective adherence to plastic (Blomhoff et al., 1984). The fractions obtained at 45 and 60ml/min, that were found to contain the majority of KC, were centrifuged at 400×g for 7min at 4°C. The resulting pellets were collected together and washed in HBSS (400×g, 7min, 4°C). The cells were then seeded on six-well plates at a density of 1–3×106/well in Dulbecco's Modified Eagle's Medium (DMEM, GibcoBRL) supplemented with 100U/ml Penicillin/Streptomycin (GibcoBRL) and 10% fetal calf serum (FCS, GibcoBRL) and incubated for 2h in a 5% CO2atmosphere at 37°C. Non-adherent cells were then removed from the dish by gently washing with fresh culture medium.

2.3 Identification of KC

KC were identified by the monoclonal antibody ED-2 (Serotec, Oxford, UK) (Barbe et al., 1990). Immunocytochemistry was performed by the indirect immunoalkaline phosphatase method on cytospins of freshly isolated cells and chamber slides of cultured cells. Briefly, slides, previously fixed with ice-cold acetone, were incubated overnight in a humidified chamber with the mouse anti-rat ED2 antibody at a dilution of 1:400. An alkaline phosphatase-conjugated rabbit anti-mouse antibody (DAKO, Glostrup, Denmark) at a dilution of 1:30 was used as the secondary antibody and the DAKO Fast Red substrate®system (DAKO) as the substrate and chromogen. Slides were lightly counterstained with hematoxylin. Cryostat sections of normal rat liver were used as controls.

Cytospins of freshly isolated cells and chamber slides of cultured cells prepared from the same experiments were also examined for the presence of non-specific esterase activity as previously described (Kolios et al., 2002). Methyl green 2% was used as counterstain.

2.4 Cell culture and treatment

Cells were cultured in DMEM with 10% FCS containing 100U/ml Penicillin/Streptomycin in a 5% CO2atmosphere at 37°C for 24h. Subsequently, the cells were cultured in the presence of various concentrations of lipopolysaccharide (LPS) from Escherichia coli (026:B6; Sigma–Aldrich, Steinheim, Germany) for 24 and 48h under serum-free conditions. The supernatant was collected and stored at −70°C until measured.

2.5 Tumor necrosis factor-a (TNF-α) and nitric oxide (NO) production

Basic and LPS-induced TNF-α production of cultured cells was measured by a commercially available solid phase enzyme-linked immunoabsorbent assay (ELISA) (Biosource, Nivelles, Belgium) according to the manufacturer's instructions. Basic and LPS-induced NO formation was determined by measuring the total nitrites/nitrates (NOx) concentration representing the end products of NO metabolism (Stamler et al., 1992). NOxwas assayed by a spectrophotometric method that uses a modification of the Griess reaction as previously described (Matrella et al., 2001).

2.6 Statistical analysis

For the analysis of differences between treatment groups and incubation times, the two-ways analysis of variance test was used. Values represent mean±SEM. Statistical significance was established at P<0.05.

3 Results and discussion

3.1 Sinusoidal cell isolation

Enzymatic digestion of liver tissue by perfusion is the first and perhaps the most crucial step of cell isolation as the final cell yield closely depends on the extent of tissue dissociation. A combination of pronase and collagenase, for enzymatic digestion, seems to give the best results (Knook et al., 1982). The use of pronase to eliminate hepatocytes is essential for the following separation steps, in order to avoid artefactual banding or cell clumping, which could reduce final purity. However, we applied minimal pronase concentration on the second digestion step to avoid extensive damage to the KC. The resulting cell suspension was found to be a heterogeneous cell population containing 30–35% ED-2 positive KC (Fig 1A).


Fig. 1

ED-2 Immunocytochemistry. (A) Cytospin preparation of the liver cell suspension before density gradient separation (200×). (B) Cells isolated from the top gradient layer. Note the majority of ED-2 positive cells (200×). (C) Interface gradient layer. Only a small portion of cells is ED-2 positive (200×). (D) Hepatocytes discarded after centrifugal elutriation (200×).


In the density gradient centrifugation step, ED-2 positive KC mixed with other sinusoidal cells were found on the top of the 11.7% gradient at a percentage of 40–60% (Fig 1B). The layer formed at the top of the 17.6% gradient contained mostly viable hepatocytes together with a small percentage of ED-2 positive KC (15–20%) (Fig 1C). These findings confirm that a significant overlap of buoyant densities exists between non-parenchymal liver cells, which sediment mainly at the top of the upper gradient. However, density gradient centrifugation is mandatory for the separation of sinusoidal cells from injured hepatocytes as pronase-damaged hepatocytes serve as ‘nuclei’ for heavy aggregation of the non-parenchymal cells in the elutriation chamber and reduce separation efficiency.

3.2 KC purification

Centrifugal elutriation was first introduced by Knook et al. (1977) to separate crude sinusoidal cell preparations based on cell size differences. Cell size has been estimated as 7μm for SC, 7.6–7.7μm for SEC and 10.2μm for KC (Vidal-Vanaclocha et al., 1993; Zahlten et al., 1978). Still, significant size overlap is observed among the non-parenchymal cells. Furthermore, significant size variation, often accompanied by functional differences, exists among cells of the same type. In this study, the sinusoidal cells obtained from the top of both gradients (11.7 and 17.6%), were introduced into the elutriation system and eluted in fractions I–IV (Table 1). The majority of non-parenchymal liver cells were eluted at fractions II, III and IV, whereas raising the flow rate further increased contamination with viable hepatocytes (Fig 3D). Interestingly, cells from all three fractions stained positively for endogenous non-specific esterase in a very high proportion (95%) (Fig. 2A–C). However, when tested with the ED2 antibody, fractions II, III and IV contained a population of 10, 60–70 and 90–95% ED2 positive cells, respectively (Fig. 3A–C). These findings suggest that endogenous esterase is not monocyte–macrophage specific, but it is present in other cell types of the liver, probably of an epithelial origin.


Table 1. Quantification of ED-2 positive cells in different elutriation fractions (2500 rpm, 25 °C)


Fig. 2

Histochemical staining for non-specific esterase. (A–C) Cytospin preparations from the elutriation fractions II, III and IV, respectively. More than 95% of cells exhibit non-specific esterase activity. (D) Cultured KC, more than 95% show non-specific esterase activity (400×).


Fig. 3

ED-2 Immunocytochemistry. (A) Cytospin preparation from the elutriation fraction II. Less than 10% of cells are ED-2 positive (400×). (B–D) Cytospin preparation from elutriation fractions III, IV, V, respectively. The majority of cells are ED-2 positive (400×).



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Overall, the fractions III and IV contained a large population of 90–120×106cells/liver, 70–80% positive for ED-2. In contrast to previous studies reporting a 90–95% purity of KC population after centrifugal elutriation (Heuff et al., 1993; Olynyk and Clarke, 1998), KC purity greater than 80%, even with elutriation flow rates slightly higher than those reported in the previous studies, was not reached. These discrepancies might be partly attributed to the fact that many previous studies apply non-specific methods to characterize the isolated cells. Many of the commonly used KC markers, such as endogenous peroxidase and non-specific esterase, phagocytosis of latex particles, and ED-1 antibodies may underestimate contamination with other non-KC sinusoidal cells (Dan and Wake, 1985; Dijkstra et al., 1985; Litwin, 1984). On the contrary, ED-2 immunocytochemistry is reported to be more specific and allows discrimination between KC and monocytes recently recruited in the liver tissue (Alric et al., 2000).

Since centrifugal elutriation is moderately effective in purifying KC, we applied the selective adherence to plastic technique as a further purification step. This resulted in a cultured population of 80–100×106cells/liver consisting almost exclusively of KC: >95% ED-2 positive cells, >95% exhibiting non-specific esterase activity (Figs. 4 and 2D). Cells attached rapidly to the dish surface, spread and finally obtained an irregular outline after the first 12h in culture (Figs. 3D and 4). Non-adherent cells removed at the final isolation step were approximately 10–15% of the seeded population, a minority (5–10%) being ED-2 positive. However, they exhibited non-specific esterase activity to a high percent (60%) and, therefore, were presumed to be mainly endothelial cells. Cells were maintained up to 14 days in culture and their ultrastructural and cytochemical characteristics remained unchanged throughout this period. Viability of cells in all separation steps was more than 98% by Trypan blue exclusion test. This is the first time that such a purity of KC population, identified by ED2 immunohistochemistry, is reported. Therefore, we believe that the application of selective adherence as a fourth isolation step after centrifugal elutriation is mandatory.


Fig. 4

ED-2 Immunocytochemistry of cultured KC. More than 95% are ED-2 positive. Cells adhere to plastic and exhibit a spread out morphology and irregular shape (400×).


3.3 Primary cultures of KC

In order to evaluate the functional capacity of the isolated cell population, KC cultures were incubated for 24 and 48h with different concentrations of LPS. This resulted in an LPS concentration-dependent production of TNF-α and NO detection in the cell culture supernatant (F=59.28, P<0.01 and F=51.45, P<0.01, respectively). We also observed a time-dependent NO release resulting in a significant difference between 24 and 48h stimulation periods (F=31.6, P<0.01). On the contrary TNF-α concentration reached a maximum plateau during the first 24h of stimulation and did not increase thereafter (Fig. 5). This finding suggests the presence of a feedback mechanism that prevents TNF-α to accumulate further.


Fig. 5

Basic and LPS-stimulated (0.1, 1, 10μg/ml) TNF-α and NO production by cultured KC. Values represent mean±SEM of three experiments.


In conclusion, effective KC isolation of high purity and yield requires a combination of low-grade enzymatic digestion with a highly selective elutriation procedure followed by selective adherence. Owing to the phenotypic variation and overlapping size and densities of non-parenchymal liver cells, we believe that taking advantage of more than one mechanism of cell separation is essential to obtain highly purified KC populations. In the present study, we describe in detail such a procedure, which gives reliable and reproducible results without altering the functional capacity of the isolated cells.

Acknowledgements

The technical assistance of Mrs K. Darivianaki is gratefully acknowledged.

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Received 28 February 2002; accepted 17 September 2002

doi:10.1016/S1065-6995(02)00249-4


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)