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Cell Biology International (2003) 27, 6773 (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
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
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.
2.1 Sinusoidal cell isolation
KC were isolated from pathogen-free male Sprague–Dawley rats over 12 months old (450–600
The organ was then detached from the perfusion device, the capsule was removed and the tissue minced to small pieces. Tissue was dispersed in 100
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% gradient. This was carefully layered onto a 17.6% Iodixanol gradient prepared by mixing 12
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 2500
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 60
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 100
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 (NO
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).
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 7
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×).
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×).
Full-size table (<1K)
Overall, the fractions III and IV contained a large population of 90–120×106
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 80–100×106
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 48
Basic and LPS-stimulated (0.1, 1, 10
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.
The technical assistance of Mrs K. Darivianaki is gratefully acknowledged.
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Received 28 February 2002; accepted 17 September 2002doi:10.1016/S1065-6995(02)00249-4