1Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Tsukuba, Japan, 2Safety Research Team, National Institute of Animal Health, Tsukuba, Japan
Kitani, H., Takenouchi, T., Sato, M., Yoshioka, M., Yamanaka, N. A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells. J. Vis. Exp. (51), e2757, doi:10.3791/2757 (2011).
Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.
1. Perfusion of the Liver
2. Preparation of Parenchymal Hepatocyte-rich Cell Fraction
3. Mixed Primary Culture of Liver Cells
4. Selective Isolation of Macrophages
5. Representative Results
An example of a mixed primary culture of adult rat liver cells is shown in Figure 2. Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells. Then, around day 6 to 12, phase contrast-bright, round macrophage-like cells vigourouly proliferate on the fibroblastic cell sheet. By shaking of the culture flasks, macrophages are readily suspended into the culture medium, and subsequent transfer and incubation in plastic dishes result in selective adhesion of macrophages(Figure 3). The macrophage-like cells can be harvested as early as day 8, and the numbers reached maximal levels on days 12 to 14. More than 106 cells can 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 per T75 culture flask (Figure 3). The purities of the isolated macrophages were 95 to 99%, as evaluated by flowcytometry or immunocytochemistry with rat macrophage-specific antibodies,such as ED-1 (Figure 4), ED-3, OX-41 (Figure 4) and Iba19.The isolated cells show typical characteristics of macrophages, such as active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
Figure 1. A scheme for selective isolation and purification of macrophage-like cells from mixed primary culture of rat liver cells.
Figure 2. Primary culture of rat liver cells and proliferation of macrophage-like cells. Arrow indicates a multinuclear giant cell. Scale bar = 100 μm. Reprinted from the Journal of Immunological Methods, Vol.360, 47-55 (2010) 9 with permission from Elsevier.
Figure 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, 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, and mitotic cells were frequently observed (arrows). Subsequent changes in cell numbers recovered from flasks at different culture periods are shown. 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. Reprinted from the Journal of Immunological Methods, Vol.360, 47-55 (2010)9 with permission from Elsevier.
Figure 4. Immunocytochemical staining of macrophage-like cells with monoclonal antibodies against rat macrophages.
Figure 5. Phagocytosis of FITC-labeled microbeads by macrophage-like cells.
Here, we report a simple and efficient method to obtain macrophages from the mixed primary cultures of adult rat liver cells. Our method relies first on the novel 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 macrophages9. It may be possible the macrophages proliferated in the mixed primary culture of adult liver cells might have originated from Kupffer or related cells that contaminated into the parenchymal hepatocytes fraction. The macrophages might have responded to specific environmental changes caused by transformation of parenchymal hepatocyte10 and other fibroblastic cells in the mixed primary cultures.
In conclusion, our method provides isolation of macrophage-like cells in sufficient number and purity from the primary culture of rat liver cellswithout using sophisticated equipment and technical skills. In addition, the isolation procedure can be repeated with the same culture flask for more than two weeks. This method might be applicable to other mammalian species.
All animals were kept and operated in the animal facility in the National Institute of Animal Health, according to the guidelines and regulations set forth by the Institution's Committee for animal experiments.
This work was funded by a research grant and a Grant-in-Aid from the Food Nanotechnology Project of the Ministry of Agriculture, Forestry, and Fisheries of Japan.
|Sodium pentobarbital solution||Kyoritsu Seiyaku||Somnopentyl Injection|
|Hank’s balanced salt solution (HBSS)||Invitrogen||14185-052|
|Ethylene glycol tetraacetic acid(EGTA)||Sigma-Aldrich||E-4378|
|Collagenase||Wako Pure Chemical Industries, Ltd.||032-10534||Cell dissociation grade
(Lot check needed)
|Eagle’s minimum essential medium (MEM)||Sigma-Aldrich||M-4655|
|Dulbecco’s modified Eagle’s medium(DMEM)||Sigma-Aldrich||D-6459||High glucose-type|
|Fetal calf serum (FCS)||Hyclone||SH30070.03||Heat inactivated at 56°C for 30 min.
(Lot check needed)
|Dulbecco’s phosphate buffered saline (PBS)||Invitrogen||14190||Ca2+-Mg2+ free|
|Î²-mercaptoethanol||Sigma-Aldrich||M-7522||Stock: 100 mM in distilled water|
|Insulin||Sigma-Aldrich||I-5500||Stock: 10 mg/ml in 0.1N HCl|
|Cell strainer||BD Biosciences||352360||Mesh size: 100 μm|
|Tissue culture flask||Sumitomo Bakelite Co., Ltd.||MS-21250||Surface area: 75 cm2|
|Non-tissue culture plastic dishes||BD Biosciences||351005|