Characterization of the liver-macrophages isolated from a mixed primary culture of neonatal swine hepatocytes.
We recently developed a novel procedure to obtain liver-macrophages in sufficient number and purity using a mixed primary culture of rat and bovine hepatocytes. In this study, we aim to apply this method to the neonatal swine liver. Swine parenchymal hepatocytes were isolated by a two-step collagenase perfusion method and cultured in T75 culture flasks. Similar to the rat and bovine cells, the swine hepatocytes retained an epithelial cell morphology for only a few days and progressively changed into fibroblastic cells. After 5-13 days of culture, macrophage-like cells actively proliferated on the mixed fibroblastic cell sheet. Gentle shaking of the culture flask followed by the transfer and brief incubation of the culture supernatant resulted in a quick and selective adhesion of macrophage-like cells to a plastic dish surface. After rinsing dishes with saline, the attached macrophage-like cells were collected at a yield of 10(6) cells per T75 culture flask at 2-3 day intervals for more than 3 weeks. The isolated cells displayed a typical macrophage morphology and were strongly positive for macrophage markers, such as CD172a, Iba-1 and KT022, but negative for cytokeratin, desmin and α-smooth muscle actin, indicating a highly purified macrophage population. The isolated cells exhibited phagocytosis of polystyrene microbeads and a release of inflammatory cytokines upon lipopolysaccharide stimulation. This shaking and attachment method is applicable to the swine liver and provides a sufficient number of macrophages without any need of complex laboratory equipments.
Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan.
Hiroshi
H
Kitani
Safety Research Team, National Institute of Animal Health, Kannondai 3-1-5, Tsukuba, Ibaraki 305-0856, Japan.
Miyako
M
Yoshioka
Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan.
Takato
T
Takenouchi
Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan.
Mitsuru
M
Sato
Safety Research Team, National Institute of Animal Health, Kannondai 3-1-5, Tsukuba, Ibaraki 305-0856, Japan.
Noriko
N
Yamanaka
Attachment; CK, cytokeratin; DAPI, 4′,6-diamidino-2-phenylindole; DES, desmin; DMEM, Dulbecco’s modified Eagle’s medium; ELISA, enzyme-linked immunosorbent assay; EMT, epithelial to mesenchymal transition; FACS, fluorescent activated cell sorter; Hepatocyte culture; Isolation; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; Macrophages; SMA, α-smooth muscle actin; Shaking; Swine