A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells

• Published in: Journal of immunological methods 2010-08, Vol.360 (1), p.47-55
• Main Authors: Kitani, Hiroshi, Takenouchi, Takato, Sato, Mitsuru, Yoshioka, Miyako, Yamanaka, Noriko
• Format: Article
• Language: English
• Subjects: Animals; Antigens, Differentiation - metabolism; Attachment; Biological and medical sciences; Cell Adhesion; Cell Culture Techniques - methods; Cell Proliferation; Cell Separation; Culture; Cytokines - metabolism; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Hepatocytes; Immunochemistry; Kupffer Cells - immunology; Kupffer Cells - metabolism; Kupffer Cells - pathology; Liver - pathology; Macrophage Activation; Macrophage-like cells; Macrophages - immunology; Macrophages - metabolism; Macrophages - pathology; Male; Molecular immunology; Phagocytosis; Proliferation; Rats; Rats, Sprague-Dawley; Shaking; Techniques
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2010.06.004

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 10 6 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.