Establishment And Characterization Of A Novel Fin Cell Line From Turbot, Scophthalmus Maximus

The turbot (Scophthalmus maximus) is a flatfish of considerable economic importance, which was reared in several countries. It has become an important aquaculture species in coastal areas of northern China since it was introduced to China. However, viral diseases with high mortalities of turbot occurred because of the intensive aquaculture. The establishment of healthy and sensitive fish cell lines is essential for isolation, identification and characterization of infectious viruses from fish. Little is known about the epidemiology of viral infections and characterization of the viruses in turbot due to lack of suitable cell lines. Thus, cell line is urgently desired in turbot for isolating and identifying viruses that cause viral diseases in this species.To initiate the in vitro culture of TF cell successfully, the fin tissue was digested with hyaluronidase (0.5%), TypeⅡcallagenase (0.2%) and trypsin (0.25%). By culturing the cells in different conditions, it was concluded that the optimal pH value and temperature for cartilage cell growth and division were pH 7.0-7.4, and temperature of 20-24℃in Leibovitz-15 medium.Primary culture of this cell was conducted at pH 7.2, 24 degrees C using Leibovitz-15 medium supplemented with antibiotics, 20% filtered bovine serum (FBS), basic fibroblast growth factor (bFGF, 10 ng/mL), epidermal growth factors (EGF, 10ng/mL), N-Acetyl-D-glucosamine(50μg/mL), and carboxymethyl—chitosan (50μg/mL). The emergence of new cell growth began from the edges of seeded tissue explants and was observed 4 weeks after the tissue explanting. The cells grew quite fast, and appeared uniformly fibroblast-like morphology. Fifty days later, the cells grew to confluence in 25 cm2 tissue culture flasks.When the TF cell grew into monolayer, they were subcultured via routine trypsin-digestion method. The TF cells were fibroblast-like and grew well enough, and have been subcultured to passage 85 and still grow in good status. The cell line has been cryopreserved in liquid nitrogen and can be recovered from storage with good cell viability. The cell morphology and growth status had no difference before and after cryopreservation.The TF cells at passage 60 were at latent stage in the first 1.5 days after subculture, and became into logarithm growing stage from day 2.5 to day 3.5. At day 4.5, the cell number was steady but the cells began to decline. The doubling time of TF cells was about 62.4 h.The chromosome number of TF cells was 44, but TF cells exhibited aneuploidy already. The number of their chromosomes varied from 28-60, of which there were 75.1% TF cells had a chromosome number of 44.Establishment of stable and normal diploid karyotype cell line from turbot will provide diagnostic and research tools for turbot biology in general, and the study of various viruses in turbot.