The cell lines from different organs of half-smooth tongue sole were the ideal material for researches on fish virology,genetic screening and functional analysis,etc.In this study,the researches of the spleen and ovary cells from female individual in vitro culture were developed by trypsin digestion method.The results showed that the spleen and ovary cells were cultured in minimum essential medium(MEM)supplemented with 20% fetal bovine serum(FBS),50 mmol/L 2-Mercaptoethanol(2-Me),100 U/mL penicillin,100 μg/mL streptomycin,1 mmol/L sodium pyruvate and 10 ng/mL basic fibroblast growth factor(bFGF),and microscopically,the cells were composed of fibroblast-like cells.The spleen cells were subcultured for 9 passages.Up to now,the ovary cells had been subcultured for 11 passages.Chromosome analysis revealed that the spleen and ovary cells maintained normal diploid number(2n=42),containing the heterogametic W chromosome.In subculture,the suitable concentration of FBS for the ovary cells was 20%.The spleen cells had been successfully transformed with red fluorescent protein(RFP)reporter plasmid.