A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells
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- 作者：Hiroshi Kitani ; Takato Takenouchi ; Mitsuru Sato ; Miyako Yoshioka ; Noriko Yamanaka
- 关键词：Macrophage-like cells ; Proliferation ; Hepatocytes ; Culture ; Shaking ; Attachment
- 刊名：Journal of Immunological Methods
- 出版年：2010
- 出版时间：31 August 2010
- 年：2010
- 卷：360
- 期：1-2
- 页码：47-55
- 全文大小：2889 K
文摘
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.