Long-term Culture of Japanese Flounder Paralichthys olivaceus Leukocytes

Abstract

We developed a long-term culture system for Japanese flounder Paralichthys olivaceus leukocytes supported by JFF07-1 feeder cells established from Japanese flounder fin tissue. Kidney leukocytes were seeded onto a monolayer of the feeder cells in enriched RDF medium supplemented with 20% fetal bovine serum and 2.5% flounder serum. Several colonies adhered to the feeder cells after 7 days of cultivation, demonstrating leukocyte proliferation. Increasing numbers of floating cells, which signified colony growth, were observed as the length of the culture period increased. The optimum culture conditions consisted of an incubation temperature of 25°C, the addition of 2.5% flounder serum to the medium and the inoculation of kidney leukocytes at a density of 2 × 106 cells/mL. The proliferated cells were grouped into three types based on May-Grunwald Giemsa staining: basophilic cytoplasmic cells (65%), neutrophilic cytoplasmic cells (30%) and large cells containing many vacuoles (5%). The cells showed acid-phosphatase activity (90%), peroxidase activity (31%) and non-specific esterase activity (57%). Electron microscopy revealed that many of the cells contained endoplasmic reticula and mitochondria, but not specific granules with the fibrillar structure that characterizes flounder granulocytes. A monocyte lineage thus appeared to be the dominant population among the proliferated cells in the culture system. The composition of growing cells was also kept after 20 passages.