- Email: [email protected]
A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells
Journal of Immunological Methods 360 (2010) 47–55
Contents lists available at ScienceDirect
Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m
Research paper
A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells Hiroshi Kitani a,⁎, Takato Takenouchi a, Mitsuru Sato a, Miyako Yoshioka b, Noriko Yamanaka b a b
Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan Safety Research Team, National Institute of Animal Health, Kannondai 3-1-5, Tsukuba, Ibaraki 305-0856, Japan
a r t i c l e
i n f o
Article history: Received 24 February 2010 Received in revised form 4 June 2010 Accepted 7 June 2010 Available online 17 June 2010 Keywords: Macrophage-like cells Proliferation Hepatocytes Culture Shaking Attachment
a b s t r a c t We report a simple and efficient method to obtain macrophage-like cells from the mixed primary cultures of adult rat liver cells. A parenchymal hepatocyte enriched fraction was prepared from adult rat livers and seeded into culture flasks. After 7 to 10 days of culture, when most hepatocytes were degenerated or transformed into fibroblastic cells, macrophage-like cells vigorously proliferated on the cell sheet. By shaking the flasks, macrophage-like cells were readily detached. Subsequent transfer and incubation in plastic dishes resulted in quick and selective adhesion of macrophage-like cells, while other contaminating cells remained suspended in the medium. After rinsing with saline, attached macrophage-like cells were harvested with 95 to 99% purity, as evaluated by flow cytometry or immunocytochemistry. These cells showed typical macrophage morphology and were strongly positive for markers of rat macrophages, such as ED-1, ED-3, and OX-41, but negative for cytokeratins and α-smooth muscle actin. They possessed functional properties of typical macrophages, including active phagocytosis of latex beads, proliferative response to recombinant GM-CSF, secretion of inflammatory and anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells. As more than 106 cells can be recovered repeatedly from a T75 culture flask at two to three day intervals for more than two weeks, our procedure might implicate a novel alternative to obtain Kupffer cells in sufficient number and purity without complex equipment and skills. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Kupffer cells (KC) are liver-specific resident macrophages and play an important role in the physiological homeostasis
Abbreviations: CK, cytokeratin; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; EMT, epithelial to mesenchymal transition; FACS, fluorescent activated cell sorter; FCS, fetal calf serum; GM-CSF, granulocyte-macrophage colony stimulating factor; IL, interleukin; KC, Kupffer cells; LPS, lipopolysaccharide; MG, microglia; PBS, phosphate buffered saline; RANTES, regulated upon activation, normal T cell expressed and secreted; SMA, α-smooth muscle actin; TNF, tumor necrosis factor; VIM, vimentin. ⁎ Corresponding author. Transgenic Animal Research Center, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki, 305-8634 Japan. Tel./fax: + 81 298 38 6043. E-mail address: [email protected] (H. Kitani). 0022-1759/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2010.06.004
of the liver (Bilzer et al., 2006; Roberts et al., 2007). KC also have important roles in acute and chronic responses of the liver to infections by bacteria and viruses and toxic or carcinogenic substances, as well as mediating hepatotoxicity (Gao et al., 2008). Activation of KC results, either directly or indirectly, in a series of inflammatory responses, such as production of specific cytokines (Ramadori and Armbrust, 2001), acute phase proteins and proteases (Fallowfield et al., 2007), and reactive oxygen species (Wu and Cederbaum, 2009). Cytokines are involved in the complex intercellular signal transduction between KC and hepatocytes, as well as intracellular signaling pathways which regulate homeostasis of liver functions (Crispe, 2009). These molecules act as protective mediators for the recovery of normal liver function but in some instances, excessive activation of KC may result in exacerbation of the damage, leading to acute and chronic liver
48
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
disease, fibrosis, or cirrhosis, and eventually failure of the organ. Proper modulation of the inflammatory activities of KC by specific reagents has been a challenge for pharmacological interventions; as a result, attempts have been made to establish and refine the isolation methods of KC from the liver. Most previous methods for KC isolation utilize dissociation of the liver cells by two-step collagenase perfusion, followed by treatment with pronase to eliminate parenchymal hepatocytes, and finally, counterflow centrifugal elutriation (Janousek et al., 1993; ten Hagen et al., 1996; Olynyk and Clarke, 1998). A number of modifications to the process have been reported, depending on the specimen and species (Heuff et al., 1994; Yoshioka et al., 1997; Alabraba et al., 2007). Although these previous methods provide certain numbers of KC with reasonable purity, they require sophisticated skills and equipment, such as a centrifugal elutriator. Furthermore, the isolation procedures for KC can only be applied once to the liver tissue from sacrificed animals. Requirement of large numbers of animals and tedious cell isolation procedures have been a major drawback of the previous methods. In this study, we provide a simple and efficient method to obtain macrophage-like cells in sufficient number and purity from mixed primary cultures of rat liver cells. 2. Materials and methods 2.1. Dissociation of liver cells and primary culture Adult male Sprague–Dawley rats (10 to 15 weeks-old) were obtained from CLEA Japan, Inc. (Tokyo, Japan) and kept in the animal facility in the National Institute of Animal Health, according to institutional guidelines for experimental animals. Parenchymal hepatocytes were isolated from the rat liver by the two-step liver perfusion method (Seglen, 1976). Parenchymal hepatocytes were suspended in growth medium composed of DMEM (high-glucose type, Invitrogen, Carlsbad, CA) containing 10% heat inactivated FCS (HyClone, Logan, UT), supplemented with 100 μM β-mercaptoethanol, 10 μg/ml insulin, 100 μg/ml streptomycin and 100 U/ml penicillin, and seeded into five to ten tissue culture flasks (surface area: 75 cm2) at a density of 6.7 × 104 cells/cm2. Three to five of these flasks were numbered for identification, and the cell numbers isolated from each flask were recorded. Culture medium was replaced every 2 to 3 days. 2.2. Isolation of macrophage-like cells by shaking and attachment After 7 to 10 days of culture, most of the hepatocytes were degenerated or transformed into fibroblastic-like cells, and macrophage-like round cells vigorously proliferated on the cell sheet. Macrophage-like cells were suspended by reciprocal shaking of the culture flasks at 80–120 strokes/min for 30– 60 min at 37 °C, and transferred into 100 mm non-tissue culture grade plastic dishes (351005, BD Biosciences, Bedford, MA). After incubation for 20–30 min at 37 °C, macrophage-like cells readily and firmly attached onto the dish surface, whereas other contaminating fibroblastic cells remained suspended in the culture medium. After rinsing with Dulbecco's PBS, attached macrophage-like cells were harvested by treating with TrypLE Express (Invitrogen), and characterized as described below.
2.3. Immunocytochemistry The isolated macrophage-like cells were seeded in eightwell chamber slides (354118, BD Biosciences) at the density of 105 cells/well with the growth medium. After 1 or 4 days of culture, the cells were fixed and immunostained as described (Takenouchi et al., 2007). The primary antibodies were as follows: mouse monoclonal anti-CD68 (ED-1, AbD Serotec, Oxford, UK); mouse monoclonal anti-CD169 (ED-3, AbD Serotec); mouse monoclonal anti-CD172a (OX-41, Millipore Co., Billerica, MA); rabbit polyclonal anti-Iba 1 (Wako Purechemical Industries, Ltd. Osaka, Japan); mouse monoclonal anti-cytokeratin 18 (CK18, CBL177, Millipore Co.); mouse monoclonal anti-cytokeratin 19 (CK19, Progen, Heidelberg, Germany); mouse monoclonal anti-α-smooth muscle actin (SMA, Progen); mouse monoclonal anti-vimentin (VIM, V-9, Sigma-Aldrich). After rinsing the slides with PBS containing 0.05% Tween 20, an EnVision system (DAKO, Hamburg, Germany) was used to visualize the antibody–antigen reaction according to the manufacturer's procedure. The immunostained slides were observed and photographed by a microscope (Model DM5000B, Leica, Bensheim, Germany) equipped with digital camera system (Model DFC480, Leica). For flow cytometry, the isolated macrophage-like cells were rinsed with PBS and fixed with 3.7% formalin in PBS at room temperature for 15 min. After permeabilization with 1% Triton X-100 in PBS for 10 min followed by blocking with 5% normal goat serum and 1% BSA in PBS for 30 min, the cells were aliquoted and incubated with primary antibodies at appropriate dilutions for 1 h at room temperature. Cells were spun down at 400 ×g for 5 min and washed with PBS containing 0.05% Tween 20. After repeating this process three times, cells were incubated with goat anti-mouse IgG labeled with Alexa Fluor 488 (Invitrogen) diluted 1:400. After incubation for 1 h at room temperature, cells were washed three times with PBS containing 0.05% Tween 20 as described. Immunolabeled cells were suspended in 0.5 ml of IsoFlow (Beckman Coulter, Fullerton, CA) and analyzed by a flow cytometer (Epics XL-MCL, Beckman Coulter). 2.4. Phagocytic assay Fluorescence-labeled polystyrene microspheres (1.0 μm diameter, Polysciences, Inc., Warrington, PA) were diluted at 1:800 in the growth medium, and added to the isolated macrophage-like cells seeded in eight-well chamber slides (105 cells/well) or 60 mm non-tissue culture grade plastic dishes (106 cells/plate). After incubation for 1, 2, and 4 h at 37 °C, cells in chamber slides were rinsed with PBS three times to remove nonphagocytosed beads (Itoh et al., 1992; Olynyk et al., 1999) and fixed with the mixture of ethanol-acetic acid as described (Takenouchi et al., 2007). The slides were covered with mounting medium containing DAPI (Vector Laboratories, Burlingame, CA), and photographed by a fluorescent microscope system equipped with digital camera (Leica). For flow cytometry, cells in plastic dishes were harvested by TrypLE Express at the time points indicated, rinsed with PBS three times to remove nonphagocytosed beads, and fixed with 3.7% formalin in PBS at room temperature for 15 min, then analyzed by a flow cytometer for phagocytosis of fluorescence-labeled microspheres.
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
49
2.5. Proliferative response to GM-CSF
3. Results
The isolated macrophage-like cells were seeded in 96-well plates (353072, BD Biosciences) in triplicate at the density of 5 × 103 cells/well with the growth medium containing recombinant GM-CSF at 3–25 ng/ml. The sources of GM-CSF used in this study were as follows: rat (G0792, Sigma-Aldrich, St. Louis, MO), mouse (G0282, Sigma-Aldrich), human (G5035, SigmaAldrich), and bovine (courtesy of Dr. Shigeki Inumaru, NIAH, Japan). After incubation for 4 days at 37 °C, cell growth was quantified by a cell viability test based on cleavage of tetrazolium salt (Cell Proliferation Reagent WST-1, Roche Diagnostics, Mannheim, Germany), according to the manufacturer's instruction. Absorbance at 440 nm was measured by a microplate reader (Molecular Devices, Sunnyvale, CA).
3.1. Primary culture of rat hepatocytes and the growth of macrophage-like cells
2.6. Cytokine production The isolated macrophage-like cells were seeded in 60 mm non-tissue culture grade plastic dishes (351007, BD Biosciences) at a density of 106 cells/plate. The next day, the medium was replaced by the growth medium containing LPS (L3129, Sigma-Aldrich) at 10 μg/ml. After incubation for 30 h at 37 °C, the culture supernatant was collected, filtered with a Millipore Millex membrane filter (0.45 μm pore size), and stored at −80 °C until use. Aliquots of samples were assayed using rat cytokine ELISA kits (Invitrogen), according to the manufacturer's instructions. The experiments were independently performed five times, and cytokine concentrations in the culture supernatant are expressed as the mean value ± SEM.
After dissociation by collagenase perfusion, followed by centrifugation at a low speed, parenchymal hepatocyte-rich cell fractions were obtained and cultured in tissue culture flasks. After incubation for one day, these cells attached on to the surface of the flasks, showing typical polygonal cobblestone like morphology with round nuclei (Fig. 1A). Binuclear cells were frequently observed. More than 95% of the cells were positive for CK18 (Fig. 2A), which is a marker for parenchymal hepatocytes. The contaminating cells detected in the cell culture were few (Fig. 2A) and included biliary epithelial cells (positive for CK19), hepatic stellate cells (positive for SMA), and fibroblastic cells (positive for VIM). Notably, small but significant numbers of ED-3 and OX-41positive cells were observed among parenchymal hepatocytes (Fig. 2A, arrows), suggesting the starting cell population contained small numbers of macrophages. These cells exhibited slender shape with some thin process, which resembled to the shape of resting macrophages in culture. Parenchymal hepatocytes lost their epithelial cell morphology within a few days in culture, and degenerated or transformed into more flattened fibroblastic cells. As the culture proceeded, around day 6, phase contrast-bright, round macrophage-like cells started to proliferate on the fibroblastic cell sheet (Fig. 1B). The growth of the macrophage-like cells continued and reached to maximum levels around day 12 (Fig. 1C) when these cells covered the flat cell
Fig. 1. Primary culture of rat liver cells and proliferation of macrophage-like cells on the fibroblastic cell sheet. After one day of culture, parenchymal hepatocytes spread on the flask's surface, and showed typical polygonal cobblestone-like morphology with one or two round nuclei (A). Parenchymal hepatocytes lost their epithelial cell morphology within a few days in culture, and transformed into more flattened fibroblastic cells. Around day 6, phase contrast-bright, round macrophage-like cells started to proliferate on the fibroblastic cell sheet (B). The growth of the macrophage-like cells continued and reached maximum levels around day 12 (C). The number of macrophage-like cells declined around day 19, when the fibroblastic cell sheet also started to degenerate (D). Colonies of macrophage-like cells occupied empty spaces of the culture flask, and multinuclear giant cells were observed (D, arrow). Scale bar = 100 μm.
50
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
Fig. 2. Immunocytochemical characterization of rat liver cells in primary culture. Cells mainly composed of parenchymal hepatocytes were obtained and seeded in eight-well chamber slides. As shown in Panel A, the culture on day 1 is mostly dominated by CK18-positive parenchymal hepatocytes, whereas the numbers of CK19-, SMA-, or VIM-positive cells were relatively small. Notably, there were few but significant numbers of slender cells which were positive for rat macrophage markers, such as ED-3 and OX-41 (arrows), suggesting the starting cell population contained small numbers of macrophages. In Panel B, on day 7, only a few CK 18positive parenchymal hepatocytes and CK 19-positive biliary epithelial cells remained in the culture, whereas SMA-positive hepatic stellate cells and VIM-positive fibroblastic cells had proliferated to form a flat cell sheet. Large numbers of ED-3 and OX-41-positive cells were observed on the flat cell sheet, indicating that macrophage-like cells were increasing in number in this specific culture environment. Scale bar = 100 μm.
sheet on the flask surface. At this stage, only a few CK18positive parenchymal hepatocytes and CK19-positive biliary epithelial cells remained in the culture (Fig. 2B), whereas SMA-positive hepatic stellate cells and VIM-positive fibro-
blastic cells had proliferated to form a flat cell sheet (Fig. 2B). Using phase contrast microscopy, large numbers of ED-3 and OX-41-positive cells were observed on the flat cell sheet (Fig. 2B), indicating macrophage-like cells were increasing in
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
51
number in this specific culture environment. To the best of our knowledge, this is the first demonstration that ordinary macrophage-like cells proliferate on the cell sheet of mixed primary cultures of rat liver cells. The number of macrophagelike cells declined to lower levels around day 19, when the fibroblastic cell sheet also started to degenerate (Fig. 1D). Colonies of macrophage-like cells occupied empty spaces of the culture flask. Multinuclear giant cells were occasionally observed (Fig. 1D, arrow). 3.2. Isolation and purification of macrophage-like cells from mixed culture flasks We sought to isolate and purify these macrophage-like cells selectively from the underlining fibroblastic cell sheet. For this purpose, we employed shaking of the culture flask, followed by selective adhesion of the cells onto non-tissue culture grade plastic dishes, as described in the Materials and methods. After shaking the flasks, macrophage-like cells were readily detached from the cell sheet. Subsequent transfer and incubation in plastic dishes resulted in quick (within 10 min) and selective adhesion of macrophage-like cells onto the dish surface (Fig. 3A), whereas contaminating fibroblastic or other cells remained suspended in the medium. After rinsing with PBS, attached macrophage-like cells were selectively harvested with high purity. These cells exhibited typical macrophage morphology 40 min after plating, and some of these cells were in mitosis, as observed under a phase contrast microscope (Fig. 3B, arrows). The average yields of the macrophage-like cells, which were repeatedly harvested from the flasks at different culture periods are shown in Fig. 3C. The macrophage-like cells can be harvested as early as day 8, and the numbers reached maximal levels on days 12 to 14. Of special note, more than 106 cells can still be harvested from a T75 culture flask repeatedly at 2 to 3 day intervals for more than two weeks, enabling a total cell yield per flask of 107. 3.3. Immunocytochemical characterization
Fig. 3. Selective isolation of macrophage-like cells by the shaking and attachment method. Cells were suspended into the culture medium by shaking the flasks, subsequently transferred into non-tissue culture grade plastic dishes, and incubated at 37 °C. As early as 10 min after plating, macrophage-like cells attached to the dish surface (A), while other contaminating fibroblastic cells remained suspended. After rinsing with PBS, a highly purified macrophage population was obtained. These cells gained typical macrophage morphology after 40 min of culture (B), and mitotic cells were frequently observed (arrows). Subsequent changes in cell numbers recovered from flasks at different culture periods is shown in C, values are an average ± SD from three to five flasks. Experiments were repeated three times and data are indicated by different symbols. Scale bar = 100 μm.
Immunocytochemistry with cell type-specific antibodies demonstrated the isolated cells were strongly positive for the markers of rat macrophages, such as ED-1, ED-3, OX-41, and Iba-1, but negative for CK18, CK19, and SMA (Fig. 4). Occasionally, a very limited number of cells positive for SMA contaminated the culture, although at a negligible proportion of b0.1%. Therefore, the purity of the rat macrophage cell population was more than 99% by immunocytochemistry. In addition, FACS analysis demonstrated more than 95% of purity of the isolated cells (Fig. 5). These cells were also strongly positive for an antibody against vimentin (Figs. 4 and 5), although the biological significance of this remains elusive. In addition, when cultured in vitro for 4 days, the macrophage-like cells fused together, forming multinuclear giant cells, as shown in the picture of ED-1 (Day 4) (Fig. 4, arrows).
FACS. The cells phagocytosed the microspheres as early as 1 h and continued until almost all cells incorporated the microspheres 4 h after administration (Fig. 6). The phagocytic activity of the cells was also quantitatively confirmed by FACS (Fig. 6), indicating the proportions of fluorescence-positive cells increased as follows; 61.6% at 1 h, 83.6% at 2 h, and 93.8% at 4 h. Although there seems to be a discrepancy between the fluorescent microscope and FACS profile at 1 h, it may be due to either the difference in culture vessels (eight-well chamber slides and 60 mm culture dishes), or the limited microscopic field (b50 cells/field) which tends to cause data variation, especially in the early stages of phagocytosis. These results demonstrated strong phagocytic activity of the isolated cells, which is a functional characteristic of macrophages, such as Kupffer cells (Itoh et al., 1992; Olynyk et al., 1999).
3.4. Phagocytic activity
3.5. Proliferative response to GM-CSF
Phagocytosis of fluorescence-labeled microspheres by the isolated cells was analyzed with a fluorescent microscope and
To further functionally characterize the isolated cells, proliferative responses to specific cytokine were studied.
52
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
Fig. 4. Immunocytochemical characterization of macrophage-like cells selectively isolated by the shaking and attachment method from mixed primary cultures of rat liver cells. The cells were plated in eight-well chamber slides in DMEM-based medium containing 10% FCS. On the next day, the cells were fixed and stained with specific antibodies against ED-1, ED-3, OX41, Iba-1, CK18, CK19, αSMA, and VIM, as described in the Materials and methods. The isolated cells were also cultured for four days, fixed, and stained with ED-1. Multinuclear giant cells were frequently observed in the culture (ED-1 day 4, arrows). Scale bar = 100 μm.
Experiments were independently repeated three times, and a typical result is shown in Fig. 7. The isolated macrophage-like cells showed strong proliferative responses to the recombinant GM-CSF from rat and mouse origin. These cells also responded to the cytokine from bovine origin at a lower degree, but not to human GM-CSF, suggesting some specificity of the cytokine among mammalian species. 3.6. Production of inflammatory and anti-inflammatory cytokines We assayed whether the isolated cells had the abilities to produce inflammatory and anti-inflammatory cytokines in response to LPS. These cells secreted significant amounts of both inflammatory (TNFα; 1639 ± 643 pg/ml, IL6; 2305 ± 160 pg/ml, IL12p40; 315 ± 74 pg/ml, and RANTES; 3075 ± 722 pg/ml) and anti-inflammatory (IL10; 511 ± 67 pg/ml) cytokines after stimulation of LPS for 30 h. In untreated negative controls, concentrations of all the cytokines examined were under the detection limit of the ELISA kits. These results demonstrated the isolated cells had abilities to produce specific cytokines in response to bacterial endotoxin.
4. Discussion Here, we report a simple and efficient method to obtain macrophage-like cells from the mixed primary cultures of adult rat liver cells. Our procedure does not require complex equipment and skills, yet provides more than 106 cells recovered repeatedly from a T75 culture flask at two to three day intervals for more than two weeks, enabling a total cell yield per flask of 107. Our method relies first on the proliferative activity of macrophages in the mixed culture of rat liver cells, and then subsequent isolation and purification of these cells on the basis of their biological characteristics as macrophages. Kupffer cells (KC) are the liver-specific resident macrophages that comprise approximately 10 to 15% of all liver cells (Hendriks et al., 1990). So, it is highly possible that the macrophage-like cells proliferated in the mixed primary culture of adult liver cells might have originated from KC contaminated to the parenchymal hepatocyte fraction. In fact, small but significant numbers of ED-3 and OX-41-positive macrophage-like cells were observed among parenchymal hepatocytes at the beginning of the culture (Fig. 2A, arrows).
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
Fig. 5. FACS analysis of the macrophage-like cells isolated by the shaking and attachment method.
Parenchymal hepatocytes, especially from rat origin, survive and maintain their morphological and physiological properties for only a limited duration in primary culture. These cells quickly degenerate or undergo phenotypic and functional conversion, which is referred to as the epithelial to mesenchymal transition (EMT). During EMT, morphological and behavioral transformations of epithelial cells are associated with the induction of specific cytoskeletal proteins for mesenchyme, such as α-smooth muscle actin and vimentin, and decreases in the level of epithelial cytokeratins (Gorrell, 2007; Zeisberg et al., 2007). We confirmed these phenotypic changes in the mixed primary cultures of rat liver cells, which resembled to those observed in fibrosis after liver injuries. A role has been suggested for macrophages in the resolution of fibrosis (Duffield et al., 2005; Fallowfield et al., 2007); therefore, it is possible macrophage-like cells responded to environmental changes caused by transformed hepatic cells during EMT and proliferated vigorously in that specific culture environment. Production of specific growth factors, such as GM-CSF or the deposition of extracellular matrix by the transformed hepatic cells might have promoted the
53
growth of macrophage-like cells. KC are considered resident macrophages in the liver (Duffield et al., 2005), but a high turnover rate of KC was shown in mouse models of bone marrow transplantation (Klein et al., 2007), suggesting their origin and continuous supply from bone-marrow precursors. These precursor cells might have contributed to the expansion of macrophage-like cells in the mixed liver cell cultures. Our method adopts, in principle, similar techniques to those used for the isolation of microglia (MG), which are well known resident macrophages in the brain (Guillemin and Brew, 2004; Sugama et al., 2009). Isolation procedures of MG have been well established (Suzumura et al., 1987; Hassan et al., 1991; Floden and Combs, 2007), as follows: mixed glial cell culture from neonatal brain cells in culture flasks, proliferation of MG on the glial cell sheet, shaking off MG from the flasks, selective attachment of MG to plastic dishes, and finally harvesting attached MG. Although this isolation procedure has been known for more than 20 years, there have been very few attempts to apply this method to other cell types in other tissues, such as KC in the liver. Before starting this study, we were involved in research on MG, such as isolation and establishment of immortalized cell lines from various mouse strains (Takenouchi et al., 2005; Iwamaru et al., 2007) and biochemical characterization of these cells, mainly on the activation of P2X7 purinergic receptor during inflammatory responses (Takenouchi et al., 2008; Takenouchi et al., 2009). When we observed the proliferation of macrophage-like cells on the cell sheet in mixed rat liver cell cultures, the morphological and physiological resemblance of these cells to MG prompted us to realize these cells might be KC. As the isolation procedure for MG using shaking and attachment method is well established and good purity is obtained, it is reasonable to assume that the cells obtained from mixed primary cultures of rat liver cells by the similar method are KC. Our method requires only a relatively small number of liver cells (5 x 106 cells per T75 flask). Nevertheless, active proliferation of macrophages in the mixed liver cell cultures, followed by selective harvest of these cells yield a large number of pure macrophages (ca. 107 in total) from a single flask during the culture period. Researchers are encouraged to apply this method to other mammalian species. We have applied this method to the bovine liver and obtained bovine macrophages with similar yield and purity as in the rat (manuscript under preparation). The applicability of the method may be particularly important for human studies, in which fresh liver samples are usually limited to those from biopsies (Heuff et al., 1994). There is heterogeneity of KC in the liver; for example, differences in their size (Armbrust and Ramadori, 1996), phagocytic activity, and the ability to produce inflammatory cytokines upon LPS stimulation (Dory et al., 2003) has been documented. Identification of these subtypes of KC has not progressed, due to the lack of molecular and biochemical markers for KC populations. Our method might enhance the opportunity to study the heterogeneity of KC by providing significant numbers of cells to evaluate cloning or immortalization of specific subtypes and their functional characterization. In conclusion, our method provides isolation of macrophagelike cells in sufficient number and purity repeatedly from the primary culture of rat liver cells. Although further studies are
54
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
Fig. 6. Phagocytosis of fluorescence-labeled microspheres by macrophage-like cells. After incubation for the indicated periods at 37 °C, cells were fixed and observed under a fluorescent microscope or analyzed by FACS, as described in the Materials and methods. Scale bar = 50 μm.
Fig. 7. Proliferative response of macrophage-like cells to GM-CSF from different mammalian species. The cells were seeded in 96-well plates in triplicate at a density of 5 × 103 cells/well with the growth medium containing recombinant GM-CSF at 3–25 ng/ml. After incubation for 4 days at 37 °C, cell growth was quantified by WST-1 cell viability test. Absorbance at 440 nm was measured by a microplate reader and mean values from 3 wells are indicated. Three independently performed experiments showed similar results.
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
necessary to compare the phenotypes of the isolated macrophages to those of KC in the liver, this method might implicate a simple and efficient alternative for the isolation of KC, without using sophisticated equipment and technical skills. Acknowledgments The authors wish to thank Dr. Jan Naessens for critical reading of the manuscript. This work was supported by a research grant and a Grant-in-Aid from the Food Nanotechnology Project of the Ministry of Agriculture, Forestry, and Fisheries of Japan. References Alabraba, E.B., Curbishley, S.M., Lai, W.K., Wigmore, S.J., Adams, D.H., Afford, S.C., 2007. A new approach to isolation and culture of human Kupffer cells. J. Immunol. Meth. 326, 139. Armbrust, T., Ramadori, G., 1996. Functional characterization of two different Kupffer cell populations of normal rat liver. J. Hepatol. 25, 518. Bilzer, M., Roggel, F., Gerbes, A.L., 2006. Role of Kupffer cells in host defense and liver disease. Liver Int. 26, 1175. Crispe, I.N., 2009. The liver as a lymphoid organ. Annu. Rev. Immunol. 27, 147. Dory, D., Echchannaoui, H., Letiembre, M., Ferracin, F., Pieters, J., Adachi, Y., Akashi, S., Zimmerli, W., Landmann, R., 2003. Generation and functional characterization of a clonal murine periportal Kupffer cell line from H-2Kb-tsA58 mice. J. Leukoc. Biol. 74, 49. Duffield, J.S., Forbes, S.J., Constandinou, C.M., Clay, S., Partolina, M., Vuthoori, S., Wu, S., Lang, R., Iredale, J.P., 2005. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J. Clin. Invest. 115, 56. Fallowfield, J.A., Mizuno, M., Kendall, T.J., Constandinou, C.M., Benyon, R.C., Duffield, J.S., Iredale, J.P., 2007. Scar-associated macrophages are a major source of hepatic matrix metalloproteinase-13 and facilitate the resolution of murine hepatic fibrosis. J. Immunol. 178, 5288. Floden, A.M., Combs, C.K., 2007. Microglia repetitively isolated from in vitro mixed glial cultures retain their initial phenotype. J. Neurosci. Meth. 164, 218. Gao, B., Jeong, W.I., Tian, Z., 2008. Liver: an organ with predominant innate immunity. Hepatology 47, 729. Gorrell, M.D., 2007. Liver fibrosis: the hepatocyte revisited. Hepatology 46, 1659. Guillemin, G.J., Brew, B.J., 2004. Microglia, macrophages, perivascular macrophages, and pericytes: a review of function and identification. J. Leukoc. Biol. 75, 388. Hassan, N.F., Rifat, S., Campbell, D.E., McCawley, L.J., Douglas, S.D., 1991. Isolation and flow cytometric characterization of newborn mouse brainderived microglia maintained in vitro. J. Leukoc. Biol. 50, 86. Hendriks, H.F., Brouwer, A., Knook, D.L., 1990. Isolation, purification, and characterization of liver cell types. Meth. Enzymol. 190, 49. Heuff, G., Meyer, S., Beelen, R.H., 1994. Isolation of rat and human Kupffer cells by a modified enzymatic assay. J. Immunol. Meth. 174, 61. Itoh, Y., Okanoue, T., Morimoto, M., Nagao, Y., Mori, T., Hori, N., Kagawa, K., Kashima, K., 1992. Functional heterogeneity of rat liver macrophages: interleukin-1 secretion and Ia antigen expression in contrast with phagocytic activity. Liver 12, 26.
55
Iwamaru, Y., Takenouchi, T., Ogihara, K., Hoshino, M., Takata, M., Imamura, M., Tagawa, Y., Hayashi-Kato, H., Ushiki-Kaku, Y., Shimizu, Y., Okada, H., Shinagawa, M., Kitani, H., Yokoyama, T., 2007. Microglial cell line established from prion protein-overexpressing mice is susceptible to various murine prion strains. J. Virol. 81, 1524. Janousek, J., Strmen, E., Gervais, F., 1993. Purification of murine Kupffer cells by centrifugal elutriation. J. Immunol. Meth. 164, 109. Klein, I., Cornejo, J.C., Polakos, N.K., John, B., Wuensch, S.A., Topham, D.J., Pierce, R.H., Crispe, I.N., 2007. Kupffer cell heterogeneity: functional properties of bone marrow derived and sessile hepatic macrophages. Blood 110, 4077. Olynyk, J.K., Britton, R.S., Stephenson, A.H., Leicester, K.L., O'Neill, R., Bacon, B.R., 1999. An in vitro model for the study of phagocytosis of damaged hepatocytes by rat Kupffer cells. Liver 19, 418. Olynyk, J.K., Clarke, S.L., 1998. Isolation and primary culture of rat Kupffer cells. J. Gastroenterol. Hepatol. 13, 842. Ramadori, G., Armbrust, T., 2001. Cytokines in the liver. Eur. J. Gastroenterol. Hepatol. 13, 777. Roberts, R.A., Ganey, P.E., Ju, C., Kamendulis, L.M., Rusyn, I., Klaunig, J.E., 2007. Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis. Toxicol. Sci. 96, 2. Seglen, P.O., 1976. Preparation of isolated rat liver cells. Meth. Cell Biol. 13, 29. Sugama, S., Takenouchi, T., Cho, B.P., Joh, T.H., Hashimoto, M., Kitani, H., 2009. Possible roles of microglial cells for neurotoxicity in clinical neurodegenerative diseases and experimental animal models. Inflamm. Allergy Drug Targets 8, 277. Suzumura, A., Mezitis, S.G., Gonatas, N.K., Silberberg, D.H., 1987. MHC antigen expression on bulk isolated macrophage-microglia from newborn mouse brain: induction of Ia antigen expression by γ-interferon. J. Neuroimmunol. 15, 263. Takenouchi, T., Iwamaru, Y., Sato, M., Yokoyama, T., Shinagawa, M., Kitani, H., 2007. Establishment and characterization of SV40 large T antigenimmortalized cell lines derived from fetal bovine brain tissues after prolonged cryopreservation. Cell Biol. Int. 31, 57. Takenouchi, T., Iwamaru, Y., Sugama, S., Sato, M., Hashimoto, M., Kitani, H., 2008. Lysophospholipids and ATP mutually suppress maturation and release of IL-1β in mouse microglial cells using a rho-dependent pathway. J. Immunol. 180, 7827. Takenouchi, T., Nakai, M., Iwamaru, Y., Sugama, S., Tsukimoto, M., Fujita, M., Wei, J., Sekigawa, A., Sato, M., Kojima, S., Kitani, H., Hashimoto, M., 2009. The activation of P2X7 receptor impairs lysosomal functions and stimulates the release of autophagolysosomes in microglial cells. J. Immunol. 182, 2051. Takenouchi, T., Ogihara, K., Sato, M., Kitani, H., 2005. Inhibitory effects of U73122 and U73343 on Ca2+ influx and pore formation induced by the activation of P2X7 nucleotide receptors in mouse microglial cell line. Biochim. Biophys. Acta 1726, 177. ten Hagen, T.L., van Vianen, W., Bakker-Woudenberg, I.A., 1996. Isolation and characterization of murine Kupffer cells and splenic macrophages. J. Immunol. Meth. 193, 81. Wu, D., Cederbaum, A.I., 2009. Oxidative stress and alcoholic liver disease. Semin. Liver Dis. 29, 141. Yoshioka, M., Nakajima, Y., Ito, T., Mikami, O., Tanaka, S., Miyazaki, S., Motoi, Y., 1997. Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells. Vet. Immunol. Immunopathol. 58, 155. Zeisberg, M., Yang, C., Martino, M., Duncan, M.B., Rieder, F., Tanjore, H., Kalluri, R., 2007. Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition. J. Biol. Chem. 282, 23337.
Contents lists available at ScienceDirect
Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m
Research paper
A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells Hiroshi Kitani a,⁎, Takato Takenouchi a, Mitsuru Sato a, Miyako Yoshioka b, Noriko Yamanaka b a b
Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan Safety Research Team, National Institute of Animal Health, Kannondai 3-1-5, Tsukuba, Ibaraki 305-0856, Japan
a r t i c l e
i n f o
Article history: Received 24 February 2010 Received in revised form 4 June 2010 Accepted 7 June 2010 Available online 17 June 2010 Keywords: Macrophage-like cells Proliferation Hepatocytes Culture Shaking Attachment
a b s t r a c t We report a simple and efficient method to obtain macrophage-like cells from the mixed primary cultures of adult rat liver cells. A parenchymal hepatocyte enriched fraction was prepared from adult rat livers and seeded into culture flasks. After 7 to 10 days of culture, when most hepatocytes were degenerated or transformed into fibroblastic cells, macrophage-like cells vigorously proliferated on the cell sheet. By shaking the flasks, macrophage-like cells were readily detached. Subsequent transfer and incubation in plastic dishes resulted in quick and selective adhesion of macrophage-like cells, while other contaminating cells remained suspended in the medium. After rinsing with saline, attached macrophage-like cells were harvested with 95 to 99% purity, as evaluated by flow cytometry or immunocytochemistry. These cells showed typical macrophage morphology and were strongly positive for markers of rat macrophages, such as ED-1, ED-3, and OX-41, but negative for cytokeratins and α-smooth muscle actin. They possessed functional properties of typical macrophages, including active phagocytosis of latex beads, proliferative response to recombinant GM-CSF, secretion of inflammatory and anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells. As more than 106 cells can be recovered repeatedly from a T75 culture flask at two to three day intervals for more than two weeks, our procedure might implicate a novel alternative to obtain Kupffer cells in sufficient number and purity without complex equipment and skills. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Kupffer cells (KC) are liver-specific resident macrophages and play an important role in the physiological homeostasis
Abbreviations: CK, cytokeratin; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; EMT, epithelial to mesenchymal transition; FACS, fluorescent activated cell sorter; FCS, fetal calf serum; GM-CSF, granulocyte-macrophage colony stimulating factor; IL, interleukin; KC, Kupffer cells; LPS, lipopolysaccharide; MG, microglia; PBS, phosphate buffered saline; RANTES, regulated upon activation, normal T cell expressed and secreted; SMA, α-smooth muscle actin; TNF, tumor necrosis factor; VIM, vimentin. ⁎ Corresponding author. Transgenic Animal Research Center, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki, 305-8634 Japan. Tel./fax: + 81 298 38 6043. E-mail address: [email protected] (H. Kitani). 0022-1759/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2010.06.004
of the liver (Bilzer et al., 2006; Roberts et al., 2007). KC also have important roles in acute and chronic responses of the liver to infections by bacteria and viruses and toxic or carcinogenic substances, as well as mediating hepatotoxicity (Gao et al., 2008). Activation of KC results, either directly or indirectly, in a series of inflammatory responses, such as production of specific cytokines (Ramadori and Armbrust, 2001), acute phase proteins and proteases (Fallowfield et al., 2007), and reactive oxygen species (Wu and Cederbaum, 2009). Cytokines are involved in the complex intercellular signal transduction between KC and hepatocytes, as well as intracellular signaling pathways which regulate homeostasis of liver functions (Crispe, 2009). These molecules act as protective mediators for the recovery of normal liver function but in some instances, excessive activation of KC may result in exacerbation of the damage, leading to acute and chronic liver
48
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
disease, fibrosis, or cirrhosis, and eventually failure of the organ. Proper modulation of the inflammatory activities of KC by specific reagents has been a challenge for pharmacological interventions; as a result, attempts have been made to establish and refine the isolation methods of KC from the liver. Most previous methods for KC isolation utilize dissociation of the liver cells by two-step collagenase perfusion, followed by treatment with pronase to eliminate parenchymal hepatocytes, and finally, counterflow centrifugal elutriation (Janousek et al., 1993; ten Hagen et al., 1996; Olynyk and Clarke, 1998). A number of modifications to the process have been reported, depending on the specimen and species (Heuff et al., 1994; Yoshioka et al., 1997; Alabraba et al., 2007). Although these previous methods provide certain numbers of KC with reasonable purity, they require sophisticated skills and equipment, such as a centrifugal elutriator. Furthermore, the isolation procedures for KC can only be applied once to the liver tissue from sacrificed animals. Requirement of large numbers of animals and tedious cell isolation procedures have been a major drawback of the previous methods. In this study, we provide a simple and efficient method to obtain macrophage-like cells in sufficient number and purity from mixed primary cultures of rat liver cells. 2. Materials and methods 2.1. Dissociation of liver cells and primary culture Adult male Sprague–Dawley rats (10 to 15 weeks-old) were obtained from CLEA Japan, Inc. (Tokyo, Japan) and kept in the animal facility in the National Institute of Animal Health, according to institutional guidelines for experimental animals. Parenchymal hepatocytes were isolated from the rat liver by the two-step liver perfusion method (Seglen, 1976). Parenchymal hepatocytes were suspended in growth medium composed of DMEM (high-glucose type, Invitrogen, Carlsbad, CA) containing 10% heat inactivated FCS (HyClone, Logan, UT), supplemented with 100 μM β-mercaptoethanol, 10 μg/ml insulin, 100 μg/ml streptomycin and 100 U/ml penicillin, and seeded into five to ten tissue culture flasks (surface area: 75 cm2) at a density of 6.7 × 104 cells/cm2. Three to five of these flasks were numbered for identification, and the cell numbers isolated from each flask were recorded. Culture medium was replaced every 2 to 3 days. 2.2. Isolation of macrophage-like cells by shaking and attachment After 7 to 10 days of culture, most of the hepatocytes were degenerated or transformed into fibroblastic-like cells, and macrophage-like round cells vigorously proliferated on the cell sheet. Macrophage-like cells were suspended by reciprocal shaking of the culture flasks at 80–120 strokes/min for 30– 60 min at 37 °C, and transferred into 100 mm non-tissue culture grade plastic dishes (351005, BD Biosciences, Bedford, MA). After incubation for 20–30 min at 37 °C, macrophage-like cells readily and firmly attached onto the dish surface, whereas other contaminating fibroblastic cells remained suspended in the culture medium. After rinsing with Dulbecco's PBS, attached macrophage-like cells were harvested by treating with TrypLE Express (Invitrogen), and characterized as described below.
2.3. Immunocytochemistry The isolated macrophage-like cells were seeded in eightwell chamber slides (354118, BD Biosciences) at the density of 105 cells/well with the growth medium. After 1 or 4 days of culture, the cells were fixed and immunostained as described (Takenouchi et al., 2007). The primary antibodies were as follows: mouse monoclonal anti-CD68 (ED-1, AbD Serotec, Oxford, UK); mouse monoclonal anti-CD169 (ED-3, AbD Serotec); mouse monoclonal anti-CD172a (OX-41, Millipore Co., Billerica, MA); rabbit polyclonal anti-Iba 1 (Wako Purechemical Industries, Ltd. Osaka, Japan); mouse monoclonal anti-cytokeratin 18 (CK18, CBL177, Millipore Co.); mouse monoclonal anti-cytokeratin 19 (CK19, Progen, Heidelberg, Germany); mouse monoclonal anti-α-smooth muscle actin (SMA, Progen); mouse monoclonal anti-vimentin (VIM, V-9, Sigma-Aldrich). After rinsing the slides with PBS containing 0.05% Tween 20, an EnVision system (DAKO, Hamburg, Germany) was used to visualize the antibody–antigen reaction according to the manufacturer's procedure. The immunostained slides were observed and photographed by a microscope (Model DM5000B, Leica, Bensheim, Germany) equipped with digital camera system (Model DFC480, Leica). For flow cytometry, the isolated macrophage-like cells were rinsed with PBS and fixed with 3.7% formalin in PBS at room temperature for 15 min. After permeabilization with 1% Triton X-100 in PBS for 10 min followed by blocking with 5% normal goat serum and 1% BSA in PBS for 30 min, the cells were aliquoted and incubated with primary antibodies at appropriate dilutions for 1 h at room temperature. Cells were spun down at 400 ×g for 5 min and washed with PBS containing 0.05% Tween 20. After repeating this process three times, cells were incubated with goat anti-mouse IgG labeled with Alexa Fluor 488 (Invitrogen) diluted 1:400. After incubation for 1 h at room temperature, cells were washed three times with PBS containing 0.05% Tween 20 as described. Immunolabeled cells were suspended in 0.5 ml of IsoFlow (Beckman Coulter, Fullerton, CA) and analyzed by a flow cytometer (Epics XL-MCL, Beckman Coulter). 2.4. Phagocytic assay Fluorescence-labeled polystyrene microspheres (1.0 μm diameter, Polysciences, Inc., Warrington, PA) were diluted at 1:800 in the growth medium, and added to the isolated macrophage-like cells seeded in eight-well chamber slides (105 cells/well) or 60 mm non-tissue culture grade plastic dishes (106 cells/plate). After incubation for 1, 2, and 4 h at 37 °C, cells in chamber slides were rinsed with PBS three times to remove nonphagocytosed beads (Itoh et al., 1992; Olynyk et al., 1999) and fixed with the mixture of ethanol-acetic acid as described (Takenouchi et al., 2007). The slides were covered with mounting medium containing DAPI (Vector Laboratories, Burlingame, CA), and photographed by a fluorescent microscope system equipped with digital camera (Leica). For flow cytometry, cells in plastic dishes were harvested by TrypLE Express at the time points indicated, rinsed with PBS three times to remove nonphagocytosed beads, and fixed with 3.7% formalin in PBS at room temperature for 15 min, then analyzed by a flow cytometer for phagocytosis of fluorescence-labeled microspheres.
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
49
2.5. Proliferative response to GM-CSF
3. Results
The isolated macrophage-like cells were seeded in 96-well plates (353072, BD Biosciences) in triplicate at the density of 5 × 103 cells/well with the growth medium containing recombinant GM-CSF at 3–25 ng/ml. The sources of GM-CSF used in this study were as follows: rat (G0792, Sigma-Aldrich, St. Louis, MO), mouse (G0282, Sigma-Aldrich), human (G5035, SigmaAldrich), and bovine (courtesy of Dr. Shigeki Inumaru, NIAH, Japan). After incubation for 4 days at 37 °C, cell growth was quantified by a cell viability test based on cleavage of tetrazolium salt (Cell Proliferation Reagent WST-1, Roche Diagnostics, Mannheim, Germany), according to the manufacturer's instruction. Absorbance at 440 nm was measured by a microplate reader (Molecular Devices, Sunnyvale, CA).
3.1. Primary culture of rat hepatocytes and the growth of macrophage-like cells
2.6. Cytokine production The isolated macrophage-like cells were seeded in 60 mm non-tissue culture grade plastic dishes (351007, BD Biosciences) at a density of 106 cells/plate. The next day, the medium was replaced by the growth medium containing LPS (L3129, Sigma-Aldrich) at 10 μg/ml. After incubation for 30 h at 37 °C, the culture supernatant was collected, filtered with a Millipore Millex membrane filter (0.45 μm pore size), and stored at −80 °C until use. Aliquots of samples were assayed using rat cytokine ELISA kits (Invitrogen), according to the manufacturer's instructions. The experiments were independently performed five times, and cytokine concentrations in the culture supernatant are expressed as the mean value ± SEM.
After dissociation by collagenase perfusion, followed by centrifugation at a low speed, parenchymal hepatocyte-rich cell fractions were obtained and cultured in tissue culture flasks. After incubation for one day, these cells attached on to the surface of the flasks, showing typical polygonal cobblestone like morphology with round nuclei (Fig. 1A). Binuclear cells were frequently observed. More than 95% of the cells were positive for CK18 (Fig. 2A), which is a marker for parenchymal hepatocytes. The contaminating cells detected in the cell culture were few (Fig. 2A) and included biliary epithelial cells (positive for CK19), hepatic stellate cells (positive for SMA), and fibroblastic cells (positive for VIM). Notably, small but significant numbers of ED-3 and OX-41positive cells were observed among parenchymal hepatocytes (Fig. 2A, arrows), suggesting the starting cell population contained small numbers of macrophages. These cells exhibited slender shape with some thin process, which resembled to the shape of resting macrophages in culture. Parenchymal hepatocytes lost their epithelial cell morphology within a few days in culture, and degenerated or transformed into more flattened fibroblastic cells. As the culture proceeded, around day 6, phase contrast-bright, round macrophage-like cells started to proliferate on the fibroblastic cell sheet (Fig. 1B). The growth of the macrophage-like cells continued and reached to maximum levels around day 12 (Fig. 1C) when these cells covered the flat cell
Fig. 1. Primary culture of rat liver cells and proliferation of macrophage-like cells on the fibroblastic cell sheet. After one day of culture, parenchymal hepatocytes spread on the flask's surface, and showed typical polygonal cobblestone-like morphology with one or two round nuclei (A). Parenchymal hepatocytes lost their epithelial cell morphology within a few days in culture, and transformed into more flattened fibroblastic cells. Around day 6, phase contrast-bright, round macrophage-like cells started to proliferate on the fibroblastic cell sheet (B). The growth of the macrophage-like cells continued and reached maximum levels around day 12 (C). The number of macrophage-like cells declined around day 19, when the fibroblastic cell sheet also started to degenerate (D). Colonies of macrophage-like cells occupied empty spaces of the culture flask, and multinuclear giant cells were observed (D, arrow). Scale bar = 100 μm.
50
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
Fig. 2. Immunocytochemical characterization of rat liver cells in primary culture. Cells mainly composed of parenchymal hepatocytes were obtained and seeded in eight-well chamber slides. As shown in Panel A, the culture on day 1 is mostly dominated by CK18-positive parenchymal hepatocytes, whereas the numbers of CK19-, SMA-, or VIM-positive cells were relatively small. Notably, there were few but significant numbers of slender cells which were positive for rat macrophage markers, such as ED-3 and OX-41 (arrows), suggesting the starting cell population contained small numbers of macrophages. In Panel B, on day 7, only a few CK 18positive parenchymal hepatocytes and CK 19-positive biliary epithelial cells remained in the culture, whereas SMA-positive hepatic stellate cells and VIM-positive fibroblastic cells had proliferated to form a flat cell sheet. Large numbers of ED-3 and OX-41-positive cells were observed on the flat cell sheet, indicating that macrophage-like cells were increasing in number in this specific culture environment. Scale bar = 100 μm.
sheet on the flask surface. At this stage, only a few CK18positive parenchymal hepatocytes and CK19-positive biliary epithelial cells remained in the culture (Fig. 2B), whereas SMA-positive hepatic stellate cells and VIM-positive fibro-
blastic cells had proliferated to form a flat cell sheet (Fig. 2B). Using phase contrast microscopy, large numbers of ED-3 and OX-41-positive cells were observed on the flat cell sheet (Fig. 2B), indicating macrophage-like cells were increasing in
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
51
number in this specific culture environment. To the best of our knowledge, this is the first demonstration that ordinary macrophage-like cells proliferate on the cell sheet of mixed primary cultures of rat liver cells. The number of macrophagelike cells declined to lower levels around day 19, when the fibroblastic cell sheet also started to degenerate (Fig. 1D). Colonies of macrophage-like cells occupied empty spaces of the culture flask. Multinuclear giant cells were occasionally observed (Fig. 1D, arrow). 3.2. Isolation and purification of macrophage-like cells from mixed culture flasks We sought to isolate and purify these macrophage-like cells selectively from the underlining fibroblastic cell sheet. For this purpose, we employed shaking of the culture flask, followed by selective adhesion of the cells onto non-tissue culture grade plastic dishes, as described in the Materials and methods. After shaking the flasks, macrophage-like cells were readily detached from the cell sheet. Subsequent transfer and incubation in plastic dishes resulted in quick (within 10 min) and selective adhesion of macrophage-like cells onto the dish surface (Fig. 3A), whereas contaminating fibroblastic or other cells remained suspended in the medium. After rinsing with PBS, attached macrophage-like cells were selectively harvested with high purity. These cells exhibited typical macrophage morphology 40 min after plating, and some of these cells were in mitosis, as observed under a phase contrast microscope (Fig. 3B, arrows). The average yields of the macrophage-like cells, which were repeatedly harvested from the flasks at different culture periods are shown in Fig. 3C. The macrophage-like cells can be harvested as early as day 8, and the numbers reached maximal levels on days 12 to 14. Of special note, more than 106 cells can still be harvested from a T75 culture flask repeatedly at 2 to 3 day intervals for more than two weeks, enabling a total cell yield per flask of 107. 3.3. Immunocytochemical characterization
Fig. 3. Selective isolation of macrophage-like cells by the shaking and attachment method. Cells were suspended into the culture medium by shaking the flasks, subsequently transferred into non-tissue culture grade plastic dishes, and incubated at 37 °C. As early as 10 min after plating, macrophage-like cells attached to the dish surface (A), while other contaminating fibroblastic cells remained suspended. After rinsing with PBS, a highly purified macrophage population was obtained. These cells gained typical macrophage morphology after 40 min of culture (B), and mitotic cells were frequently observed (arrows). Subsequent changes in cell numbers recovered from flasks at different culture periods is shown in C, values are an average ± SD from three to five flasks. Experiments were repeated three times and data are indicated by different symbols. Scale bar = 100 μm.
Immunocytochemistry with cell type-specific antibodies demonstrated the isolated cells were strongly positive for the markers of rat macrophages, such as ED-1, ED-3, OX-41, and Iba-1, but negative for CK18, CK19, and SMA (Fig. 4). Occasionally, a very limited number of cells positive for SMA contaminated the culture, although at a negligible proportion of b0.1%. Therefore, the purity of the rat macrophage cell population was more than 99% by immunocytochemistry. In addition, FACS analysis demonstrated more than 95% of purity of the isolated cells (Fig. 5). These cells were also strongly positive for an antibody against vimentin (Figs. 4 and 5), although the biological significance of this remains elusive. In addition, when cultured in vitro for 4 days, the macrophage-like cells fused together, forming multinuclear giant cells, as shown in the picture of ED-1 (Day 4) (Fig. 4, arrows).
FACS. The cells phagocytosed the microspheres as early as 1 h and continued until almost all cells incorporated the microspheres 4 h after administration (Fig. 6). The phagocytic activity of the cells was also quantitatively confirmed by FACS (Fig. 6), indicating the proportions of fluorescence-positive cells increased as follows; 61.6% at 1 h, 83.6% at 2 h, and 93.8% at 4 h. Although there seems to be a discrepancy between the fluorescent microscope and FACS profile at 1 h, it may be due to either the difference in culture vessels (eight-well chamber slides and 60 mm culture dishes), or the limited microscopic field (b50 cells/field) which tends to cause data variation, especially in the early stages of phagocytosis. These results demonstrated strong phagocytic activity of the isolated cells, which is a functional characteristic of macrophages, such as Kupffer cells (Itoh et al., 1992; Olynyk et al., 1999).
3.4. Phagocytic activity
3.5. Proliferative response to GM-CSF
Phagocytosis of fluorescence-labeled microspheres by the isolated cells was analyzed with a fluorescent microscope and
To further functionally characterize the isolated cells, proliferative responses to specific cytokine were studied.
52
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
Fig. 4. Immunocytochemical characterization of macrophage-like cells selectively isolated by the shaking and attachment method from mixed primary cultures of rat liver cells. The cells were plated in eight-well chamber slides in DMEM-based medium containing 10% FCS. On the next day, the cells were fixed and stained with specific antibodies against ED-1, ED-3, OX41, Iba-1, CK18, CK19, αSMA, and VIM, as described in the Materials and methods. The isolated cells were also cultured for four days, fixed, and stained with ED-1. Multinuclear giant cells were frequently observed in the culture (ED-1 day 4, arrows). Scale bar = 100 μm.
Experiments were independently repeated three times, and a typical result is shown in Fig. 7. The isolated macrophage-like cells showed strong proliferative responses to the recombinant GM-CSF from rat and mouse origin. These cells also responded to the cytokine from bovine origin at a lower degree, but not to human GM-CSF, suggesting some specificity of the cytokine among mammalian species. 3.6. Production of inflammatory and anti-inflammatory cytokines We assayed whether the isolated cells had the abilities to produce inflammatory and anti-inflammatory cytokines in response to LPS. These cells secreted significant amounts of both inflammatory (TNFα; 1639 ± 643 pg/ml, IL6; 2305 ± 160 pg/ml, IL12p40; 315 ± 74 pg/ml, and RANTES; 3075 ± 722 pg/ml) and anti-inflammatory (IL10; 511 ± 67 pg/ml) cytokines after stimulation of LPS for 30 h. In untreated negative controls, concentrations of all the cytokines examined were under the detection limit of the ELISA kits. These results demonstrated the isolated cells had abilities to produce specific cytokines in response to bacterial endotoxin.
4. Discussion Here, we report a simple and efficient method to obtain macrophage-like cells from the mixed primary cultures of adult rat liver cells. Our procedure does not require complex equipment and skills, yet provides more than 106 cells recovered repeatedly from a T75 culture flask at two to three day intervals for more than two weeks, enabling a total cell yield per flask of 107. Our method relies first on the proliferative activity of macrophages in the mixed culture of rat liver cells, and then subsequent isolation and purification of these cells on the basis of their biological characteristics as macrophages. Kupffer cells (KC) are the liver-specific resident macrophages that comprise approximately 10 to 15% of all liver cells (Hendriks et al., 1990). So, it is highly possible that the macrophage-like cells proliferated in the mixed primary culture of adult liver cells might have originated from KC contaminated to the parenchymal hepatocyte fraction. In fact, small but significant numbers of ED-3 and OX-41-positive macrophage-like cells were observed among parenchymal hepatocytes at the beginning of the culture (Fig. 2A, arrows).
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
Fig. 5. FACS analysis of the macrophage-like cells isolated by the shaking and attachment method.
Parenchymal hepatocytes, especially from rat origin, survive and maintain their morphological and physiological properties for only a limited duration in primary culture. These cells quickly degenerate or undergo phenotypic and functional conversion, which is referred to as the epithelial to mesenchymal transition (EMT). During EMT, morphological and behavioral transformations of epithelial cells are associated with the induction of specific cytoskeletal proteins for mesenchyme, such as α-smooth muscle actin and vimentin, and decreases in the level of epithelial cytokeratins (Gorrell, 2007; Zeisberg et al., 2007). We confirmed these phenotypic changes in the mixed primary cultures of rat liver cells, which resembled to those observed in fibrosis after liver injuries. A role has been suggested for macrophages in the resolution of fibrosis (Duffield et al., 2005; Fallowfield et al., 2007); therefore, it is possible macrophage-like cells responded to environmental changes caused by transformed hepatic cells during EMT and proliferated vigorously in that specific culture environment. Production of specific growth factors, such as GM-CSF or the deposition of extracellular matrix by the transformed hepatic cells might have promoted the
53
growth of macrophage-like cells. KC are considered resident macrophages in the liver (Duffield et al., 2005), but a high turnover rate of KC was shown in mouse models of bone marrow transplantation (Klein et al., 2007), suggesting their origin and continuous supply from bone-marrow precursors. These precursor cells might have contributed to the expansion of macrophage-like cells in the mixed liver cell cultures. Our method adopts, in principle, similar techniques to those used for the isolation of microglia (MG), which are well known resident macrophages in the brain (Guillemin and Brew, 2004; Sugama et al., 2009). Isolation procedures of MG have been well established (Suzumura et al., 1987; Hassan et al., 1991; Floden and Combs, 2007), as follows: mixed glial cell culture from neonatal brain cells in culture flasks, proliferation of MG on the glial cell sheet, shaking off MG from the flasks, selective attachment of MG to plastic dishes, and finally harvesting attached MG. Although this isolation procedure has been known for more than 20 years, there have been very few attempts to apply this method to other cell types in other tissues, such as KC in the liver. Before starting this study, we were involved in research on MG, such as isolation and establishment of immortalized cell lines from various mouse strains (Takenouchi et al., 2005; Iwamaru et al., 2007) and biochemical characterization of these cells, mainly on the activation of P2X7 purinergic receptor during inflammatory responses (Takenouchi et al., 2008; Takenouchi et al., 2009). When we observed the proliferation of macrophage-like cells on the cell sheet in mixed rat liver cell cultures, the morphological and physiological resemblance of these cells to MG prompted us to realize these cells might be KC. As the isolation procedure for MG using shaking and attachment method is well established and good purity is obtained, it is reasonable to assume that the cells obtained from mixed primary cultures of rat liver cells by the similar method are KC. Our method requires only a relatively small number of liver cells (5 x 106 cells per T75 flask). Nevertheless, active proliferation of macrophages in the mixed liver cell cultures, followed by selective harvest of these cells yield a large number of pure macrophages (ca. 107 in total) from a single flask during the culture period. Researchers are encouraged to apply this method to other mammalian species. We have applied this method to the bovine liver and obtained bovine macrophages with similar yield and purity as in the rat (manuscript under preparation). The applicability of the method may be particularly important for human studies, in which fresh liver samples are usually limited to those from biopsies (Heuff et al., 1994). There is heterogeneity of KC in the liver; for example, differences in their size (Armbrust and Ramadori, 1996), phagocytic activity, and the ability to produce inflammatory cytokines upon LPS stimulation (Dory et al., 2003) has been documented. Identification of these subtypes of KC has not progressed, due to the lack of molecular and biochemical markers for KC populations. Our method might enhance the opportunity to study the heterogeneity of KC by providing significant numbers of cells to evaluate cloning or immortalization of specific subtypes and their functional characterization. In conclusion, our method provides isolation of macrophagelike cells in sufficient number and purity repeatedly from the primary culture of rat liver cells. Although further studies are
54
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
Fig. 6. Phagocytosis of fluorescence-labeled microspheres by macrophage-like cells. After incubation for the indicated periods at 37 °C, cells were fixed and observed under a fluorescent microscope or analyzed by FACS, as described in the Materials and methods. Scale bar = 50 μm.
Fig. 7. Proliferative response of macrophage-like cells to GM-CSF from different mammalian species. The cells were seeded in 96-well plates in triplicate at a density of 5 × 103 cells/well with the growth medium containing recombinant GM-CSF at 3–25 ng/ml. After incubation for 4 days at 37 °C, cell growth was quantified by WST-1 cell viability test. Absorbance at 440 nm was measured by a microplate reader and mean values from 3 wells are indicated. Three independently performed experiments showed similar results.
H. Kitani et al. / Journal of Immunological Methods 360 (2010) 47–55
necessary to compare the phenotypes of the isolated macrophages to those of KC in the liver, this method might implicate a simple and efficient alternative for the isolation of KC, without using sophisticated equipment and technical skills. Acknowledgments The authors wish to thank Dr. Jan Naessens for critical reading of the manuscript. This work was supported by a research grant and a Grant-in-Aid from the Food Nanotechnology Project of the Ministry of Agriculture, Forestry, and Fisheries of Japan. References Alabraba, E.B., Curbishley, S.M., Lai, W.K., Wigmore, S.J., Adams, D.H., Afford, S.C., 2007. A new approach to isolation and culture of human Kupffer cells. J. Immunol. Meth. 326, 139. Armbrust, T., Ramadori, G., 1996. Functional characterization of two different Kupffer cell populations of normal rat liver. J. Hepatol. 25, 518. Bilzer, M., Roggel, F., Gerbes, A.L., 2006. Role of Kupffer cells in host defense and liver disease. Liver Int. 26, 1175. Crispe, I.N., 2009. The liver as a lymphoid organ. Annu. Rev. Immunol. 27, 147. Dory, D., Echchannaoui, H., Letiembre, M., Ferracin, F., Pieters, J., Adachi, Y., Akashi, S., Zimmerli, W., Landmann, R., 2003. Generation and functional characterization of a clonal murine periportal Kupffer cell line from H-2Kb-tsA58 mice. J. Leukoc. Biol. 74, 49. Duffield, J.S., Forbes, S.J., Constandinou, C.M., Clay, S., Partolina, M., Vuthoori, S., Wu, S., Lang, R., Iredale, J.P., 2005. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J. Clin. Invest. 115, 56. Fallowfield, J.A., Mizuno, M., Kendall, T.J., Constandinou, C.M., Benyon, R.C., Duffield, J.S., Iredale, J.P., 2007. Scar-associated macrophages are a major source of hepatic matrix metalloproteinase-13 and facilitate the resolution of murine hepatic fibrosis. J. Immunol. 178, 5288. Floden, A.M., Combs, C.K., 2007. Microglia repetitively isolated from in vitro mixed glial cultures retain their initial phenotype. J. Neurosci. Meth. 164, 218. Gao, B., Jeong, W.I., Tian, Z., 2008. Liver: an organ with predominant innate immunity. Hepatology 47, 729. Gorrell, M.D., 2007. Liver fibrosis: the hepatocyte revisited. Hepatology 46, 1659. Guillemin, G.J., Brew, B.J., 2004. Microglia, macrophages, perivascular macrophages, and pericytes: a review of function and identification. J. Leukoc. Biol. 75, 388. Hassan, N.F., Rifat, S., Campbell, D.E., McCawley, L.J., Douglas, S.D., 1991. Isolation and flow cytometric characterization of newborn mouse brainderived microglia maintained in vitro. J. Leukoc. Biol. 50, 86. Hendriks, H.F., Brouwer, A., Knook, D.L., 1990. Isolation, purification, and characterization of liver cell types. Meth. Enzymol. 190, 49. Heuff, G., Meyer, S., Beelen, R.H., 1994. Isolation of rat and human Kupffer cells by a modified enzymatic assay. J. Immunol. Meth. 174, 61. Itoh, Y., Okanoue, T., Morimoto, M., Nagao, Y., Mori, T., Hori, N., Kagawa, K., Kashima, K., 1992. Functional heterogeneity of rat liver macrophages: interleukin-1 secretion and Ia antigen expression in contrast with phagocytic activity. Liver 12, 26.
55
Iwamaru, Y., Takenouchi, T., Ogihara, K., Hoshino, M., Takata, M., Imamura, M., Tagawa, Y., Hayashi-Kato, H., Ushiki-Kaku, Y., Shimizu, Y., Okada, H., Shinagawa, M., Kitani, H., Yokoyama, T., 2007. Microglial cell line established from prion protein-overexpressing mice is susceptible to various murine prion strains. J. Virol. 81, 1524. Janousek, J., Strmen, E., Gervais, F., 1993. Purification of murine Kupffer cells by centrifugal elutriation. J. Immunol. Meth. 164, 109. Klein, I., Cornejo, J.C., Polakos, N.K., John, B., Wuensch, S.A., Topham, D.J., Pierce, R.H., Crispe, I.N., 2007. Kupffer cell heterogeneity: functional properties of bone marrow derived and sessile hepatic macrophages. Blood 110, 4077. Olynyk, J.K., Britton, R.S., Stephenson, A.H., Leicester, K.L., O'Neill, R., Bacon, B.R., 1999. An in vitro model for the study of phagocytosis of damaged hepatocytes by rat Kupffer cells. Liver 19, 418. Olynyk, J.K., Clarke, S.L., 1998. Isolation and primary culture of rat Kupffer cells. J. Gastroenterol. Hepatol. 13, 842. Ramadori, G., Armbrust, T., 2001. Cytokines in the liver. Eur. J. Gastroenterol. Hepatol. 13, 777. Roberts, R.A., Ganey, P.E., Ju, C., Kamendulis, L.M., Rusyn, I., Klaunig, J.E., 2007. Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis. Toxicol. Sci. 96, 2. Seglen, P.O., 1976. Preparation of isolated rat liver cells. Meth. Cell Biol. 13, 29. Sugama, S., Takenouchi, T., Cho, B.P., Joh, T.H., Hashimoto, M., Kitani, H., 2009. Possible roles of microglial cells for neurotoxicity in clinical neurodegenerative diseases and experimental animal models. Inflamm. Allergy Drug Targets 8, 277. Suzumura, A., Mezitis, S.G., Gonatas, N.K., Silberberg, D.H., 1987. MHC antigen expression on bulk isolated macrophage-microglia from newborn mouse brain: induction of Ia antigen expression by γ-interferon. J. Neuroimmunol. 15, 263. Takenouchi, T., Iwamaru, Y., Sato, M., Yokoyama, T., Shinagawa, M., Kitani, H., 2007. Establishment and characterization of SV40 large T antigenimmortalized cell lines derived from fetal bovine brain tissues after prolonged cryopreservation. Cell Biol. Int. 31, 57. Takenouchi, T., Iwamaru, Y., Sugama, S., Sato, M., Hashimoto, M., Kitani, H., 2008. Lysophospholipids and ATP mutually suppress maturation and release of IL-1β in mouse microglial cells using a rho-dependent pathway. J. Immunol. 180, 7827. Takenouchi, T., Nakai, M., Iwamaru, Y., Sugama, S., Tsukimoto, M., Fujita, M., Wei, J., Sekigawa, A., Sato, M., Kojima, S., Kitani, H., Hashimoto, M., 2009. The activation of P2X7 receptor impairs lysosomal functions and stimulates the release of autophagolysosomes in microglial cells. J. Immunol. 182, 2051. Takenouchi, T., Ogihara, K., Sato, M., Kitani, H., 2005. Inhibitory effects of U73122 and U73343 on Ca2+ influx and pore formation induced by the activation of P2X7 nucleotide receptors in mouse microglial cell line. Biochim. Biophys. Acta 1726, 177. ten Hagen, T.L., van Vianen, W., Bakker-Woudenberg, I.A., 1996. Isolation and characterization of murine Kupffer cells and splenic macrophages. J. Immunol. Meth. 193, 81. Wu, D., Cederbaum, A.I., 2009. Oxidative stress and alcoholic liver disease. Semin. Liver Dis. 29, 141. Yoshioka, M., Nakajima, Y., Ito, T., Mikami, O., Tanaka, S., Miyazaki, S., Motoi, Y., 1997. Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells. Vet. Immunol. Immunopathol. 58, 155. Zeisberg, M., Yang, C., Martino, M., Duncan, M.B., Rieder, F., Tanjore, H., Kalluri, R., 2007. Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition. J. Biol. Chem. 282, 23337.