A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells.
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 alpha-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.
Kitani
Hiroshi
H
Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan. kitani@affrc.go.jp
Takenouchi
Takato
T
Sato
Mitsuru
M
Yoshioka
Miyako
M
Yamanaka
Noriko
N
D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism*; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation; D002469:Cell Separation; D016207:Cytokines / Q000378:metabolism; D005434:Flow Cytometry; D007120:Immunochemistry; D007728:Kupffer Cells / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D008099:Liver / Q000473:pathology*; D008262:Macrophage Activation; D008264:Macrophages / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D008297:Male; D010587:Phagocytosis; D051381:Rats; D017207:Rats, Sprague-Dawley
D000943:Antigens, Differentiation; D016207:Cytokines