Abstract：The development of fish cell line provides an important tool to study virology, immunology, genetics, oncology, developmental biology, toxicology, clinical medicine and biotechnology in aquaculture. Many fish cell lines have been established from freshwater and anadromous species, but few in marine fishes. As a widely cultivated marine fish species with high economic value in Europe and China, the number of cell lines was inadequacies in the research of turbot (Scophthalmus maximus). To expand quantity of marine fish cell lines, also provide alternative tool for gene function research or the isolation of the virus, a new cell line, Scophthalmus maximus heart cell line (SMH), was established from turbot heart in the research. Tissue block method was used and the heart tissues were collected aseptically, and washed 3 times with phosphate-buffered saline (PBS) and minced into small pieces (1 mm3) by surgical scissors in DMEM-F12 medium (pH 7.2). Then tissue pieces were washed again with PBS until it was neat without blood. It had been cultured in 25 cm2 cell-culture flask which should be inverted at 24 ℃, then returned the flask 4~5 h later. After completion of the above steps, 3 mL DMEM-F12 complete medium was added into the flask, which was supplemented with 20% FBS, 50 mmol/L 2- mercaptoethanol (2-Me), 10 ng/mL basic fibroblast growth factor (bFGF), 100 IU/mL penicillin and 100 μg/mL streptomycin, 20 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (hepes). When cells grew into a conflent monolayer, they were subcultured by trypsin solution (0.25% trypsin and 0.03% EDTA in PBS) at a split ratio of 1∶2. The cultured SMH cells, fibroblastic in morphology, had been subcultured to passage 58 in 230 d in a good proliferating state. The SMH cells at passage 23 were cryopreserved in liquid nitrogen. After 3 months, the cells could undergo cryopreservation with proliferated to 80% confluency in 5~6 d. The morphology and proliferation ability of SMH cells were the same before and after cryopreservation after thawing. The growth curve of SMH cells at passage 30 suggested that the SMH cells were at latent stage on the beginning of 2 d and went into logarithmic stage of 3~4 d. And their doubling time was calculated to be 41.28 h at passage 30. Transfection experiment demonstrated that SMH cells transfected with pEGFP-N3 plasmid could express green florescence protein (GFP) with higher transfection efficiency. The green florescence signals were observed every 12 h under a Nikon Eclipse T2000-U florescence microscope. GFP expressed successfully in cells and achieved maximum fluorescence value at 96 h. Using geneticin (G418) to screen SMH cells which transfected by recombinant plasmid, showed a bunch of cells with higher efficiency of fluorescence. Chromosome analysis revealed that the SMH cell line had a normal diploid karyotype with 2n=44, which occupied 64% in the 100 metaphase cells counted at passage 42. The SMH cells sequence of cytochrome oxidase subunitⅠ(COⅠ) gene showed a 99% consistency with turbot (GenBank Accession No. KJ205427). Above all, the species of SMH could be identified from turbot by mitochondrial DNA COⅠ gene. The establishment of the SMH can provide experimental material for genetic research capabilities or more, and importantly, could progress the study of this cell line in virus isolation and cell-pathogen interaction study as more fish virus susceptibility to SMH cell line needing to be detected.
