Isolation, cultivation and identification of Ctenopharyngodon idella intestinal macrophages

In order to establish the method for primary isolation, culture and identification of grass carp (Ctenopharyngodon idella) intestinal macrophages, in the study, the intestinal mucosal lamina propria mononuclear cells (LPMC) were firstly prepared by curettage combined with collagenase IV digestion. Then, the intestinal macrophages were isolated from LPMC suspension using fish organ mononuclear cell isolation kit and purified by differential adherence method. The intestinal macrophages after purification were cultured primarily with RPMI 1640 complete medium supplemented with 10% FBS and 5% serum of C. idella at 28 °C in a 5% CO₂ incubator, and these cells were identified by morphological examination, molecular marker detection and function validation test. The results showed that the yield of intestinal macrophages was about 3×10⁷ cells per fish (about 250 g in weight), with a survival rate of 99.6% and a purity of more than 95%, respectively. The adherent cells in primary culture for 4–5 days were round or polygonal, the cell volume increased obviously, and the confluence degree of cell layer reached 95% observed under inverted microscope. After staining, the adherent cells had the morphological and structural characteristics of macrophages observed under light and electron microscopes, respectively. The expression of macrophage-specific marker molecule (macrophage colony stimulating factor receptor of C. idella) was detected by RT-PCR. It was also confirmed that the adherent cells in primary culture had phagocytosis, and LPS could significantly improve their respiratory burst activity. The above results indicated that the method for isolation, primary culture and identification of C. idella intestinal macrophages was successfully established for the first time, which might provide a cell model for research on the intestinal mucosal immune response of C. idella induced with oral vaccine.