This suggests that the parenchymal hepatocyte-rich cell fraction SCH727965 manufacturer contained a small but nevertheless substantial number of macrophage-related cells. As the culture proceeded from day 5–13, the parenchymal hepatocytes lost the epithelial cell morphology and morphologically turned into more flattened, fibroblast-like cells (Fig. 1). Some of these transformed

hepatocytes were positive for SMA (Fig. 2B) around day 3, although most of the other cells still continued to express CK18 (Fig. 2B). The number of SMA-positive cells increased as the culture proceeded further after 9 days of culture (Fig. 2C). These results suggest that the epithelial to mesenchymal transition (EMT) was progressively induced in swine hepatocytes under these culture conditions, and the SMA-positive hepatic stellate or smooth muscle cells became dominant. Concomitantly, phase contrast-bright, round macrophage-like cells started to proliferate on the fibroblastic Erastin chemical structure cell sheet around day 9 (Fig. 1 and Fig. 2, arrowheads). The growth of the macrophage-like cells continued and reached a plateau around day 13

and thereafter (Fig. 1). In accordance with the phase contrast microscopy, immunocytochemistry demonstrated many CD172a-positive cells on the fibroblastic cell sheet (Fig. 2C). Therefore, as observed in the rat [12] and bovine [14] livers, macrophage-like cells actively proliferated on a cell sheet having a mixed primary culture of swine hepatocytes. Macrophage-like cells were suspended in the culture medium by shaking the culture flasks, followed by transfer into plastic dishes. After incubation for 6 h at 37 °C, macrophage-like cells readily became attached to the dish surface (Fig. 3A), whereas fibroblastic cells or other cells remained suspended in the medium. These Niclosamide contaminating cells were removed by washing with PBS. After 24 h culture, these cells exhibited a typical macrophage-like

morphology, extending filopodia and lamellipodia (Fig. 3B). The average yields of the macrophage-like cells, which were repeatedly harvested from the flasks at different culture periods, are shown in Fig. 3C. The macrophage-like cells were harvested as early as day 9; thereafter, more than 106 cells were harvested from each T75 flask repeatedly at 2–3 day intervals for at least 3 weeks or more. These results demonstrate that the shaking and attachment method is applicable to primary cultures of swine liver cells, with a total macrophage-like cell yield per flask of more than 107. Immunocytochemsitry with cell type-specific antibodies demonstrated that almost all the isolated cells were strongly positive for macrophage markers, such as CD172a, Iba-1 and KT022, but negative for CK18, SMA and DES (Fig. 4A).