A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.
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.
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.
The authors have nothing to disclose.
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.
Name of the reagent | Company | Catalogue number | Comments (optional) |
---|---|---|---|
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) |
Trypsin inhibitor | Sigma-Aldrich | T-9128 | |
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) |
TrypLE Express | Invitrogen | 12605 | |
Dulbecco’s phosphate buffered saline (PBS) | Invitrogen | 14190 | Ca2+-Mg2+ free |
β-mercaptoethanol | Sigma-Aldrich | M-7522 | Stock: 100 mM in distilled water |
Insulin | Sigma-Aldrich | I-5500 | Stock: 10 mg/ml in 0.1N HCl |
Penicillin/ streptomycin | Invitrogen | 15070 | |
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 | |
Cell scraper | Corning Inc. | 3010 | |
Reciprocal shaker | TAITEC | NR-1 |