Characterization of the liver-macrophages isolated from a mixed primary culture of neonatal swine hepatocytes

Abstract

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 106 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 a-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.

Establishment of SV40 large T antigen‐immortalized bovine liver sinusoidal cell lines and their immunological responses to deoxynivalenol and lipopolysaccharide

“…The proportion of contaminating cells that were positive for a-SMA was less than 1% by immunocytochemistry, indicating the purity of the cell population as Kupffer cells to be more than 99% (data not shown). The purity of the isolated Kupffer cells was comparable to that in the previous report (Kitani et al, 2011(Kitani et al, , 2014.…”

“…For the immunocytochemical characterization, we used a series of antibodies that detect cell-type specific marker proteins of liver cells as described in Table 1. Primary BKC exhibited a macrophage-like morphology, as previously reported by Kitani et al (2011Kitani et al ( , 2014. In addition, the isolated BKC were strongly positive for CD11b, CD172a, and MHC-II; weakly positive for CD14 and CD68; but negative for CD11c ( Figure 1A).…”

“…Bovine Kupffer cells (BKC) have been isolated by the counterflow centrifugal elutriation method, and these BKC expressed mRNAs of TNF‐α, IL‐1α, IL‐1β, and IL‐6 by stimulation of LPS (Yoshioka et al, ). Recently, we have reported a simple and efficient procedure to obtain Kupffer cells in sufficient numbers and purity using a mixed primary culture of hepatocytes from rat, bovine, and porcine species (Kitani et al, , , ).…”

Macrophage polarization in response to epigenetic modifiers during infection and inflammation

“…These cells represent approximately 10-15% of all liver cells. Kupffer cells attack blood-borne particles, whereas other macrophages serve in immune surveillance [28]. Dermal macrophages and Langerhans cells take up residence in the skin.…”

Extracellular ATP does not induce P2X7 receptor-dependent responses in cultured renal- and liver-derived swine macrophages

“…A previous study showed the functional expression of P2X7R in swine ovarian theca cells [9] . In this study, to understand the role of P2X7R in the innate immune defense of swine, we aim to study the expression and function of P2X7R in swine macrophages, which were obtained from mixed primary cultures of swine kidney or liver tissues [10] . Despite the presence of P2X7R mRNA and protein, extracellular ATP failed to induce P2X7R-dependent cellular responses in the swine macrophages.…”