Isolation, culture, and characterization of macrophages from the head kidney of Japanese flounder (Paralichthys olivaceus)

Abstract:

The innate immune system of the Japanese flounder plays a vital role in resisting the invasion of pathogens. A macrophage is a type of leukocyte found in body tissues, which are part of the innate immune system. Macrophages are crucial to the immune response and play an important role in the clearance and phagocytosis of pathogens and abnormal cells. Macrophages can not be subcultured, so it is necessary to establish an efficient technique for macrophage isolation and culture. In this study, macrophages were isolated from the head kidney of the Japanese flounder, and the obtained cells were purified by differential adherence. Trypan blue staining showed that the cell survival rate was 99.62%. Subsequently, L-15 medium (Gibco), 5% fetal bovine serum, 1% mycillin, 1% nonessential amino acids, and 30% L929 cell culture medium were used to culture the cells at 24℃. We compared the macrophages of Japanese flounder from different culture media and different serum concentrations. The results showed that the L-15 medium (Gibco) and 5% serum culture were the best. Cells cultured under this condition had a higher adherence rate, remaining above 85% after seven days of culture. Microscopic observations and Giemsa staining showed that the adherent cells had similar morphological characteristics to the macrophages, including a round shape and oval cell nuclei that were biased toward the side of the cell. Cells were identified via the macrophage-specific marker mpeg1, and the results showed that the gene was successfully amplified in the isolated cells. In this study, techniques to isolate and culture Japanese flounder macrophages were established, providing a basis to study macrophage functioning and to better understand the innate immunity of teleost fish in vitro.