Establishment And Characterization Of Four Cell Lines Derived From European Eel And Large Yellow Croaker

At present, fish farming is facing a series of problems such as germplasm degeneration,environmental degradation, and more frequent outbreaks of the disease. In order to solve these issues, relevant toxicology, pharmacology, vaccine, genetics and breeding of fish must be further studied. Fish cell lines cultured in vitro provide us an economical, better repeatable, efficient way which can effectively avoid individual differences and operational disadvantages caused by the high mortality rate of fish cultured in lab environment. Development and characterization of fish cell lines is the key to achieve the above mentioned conveniences.In this study, classical in vitro fish tissue culture method with optimized media and culture conditions to accommodate different fish species and ages was employed. Finally, 4 cell lines from of European eel’s(Anguilla anguilla) caudal fin and large yellow croaker’s(Larimichthys crocea) brain tissue, liver tissue and muscle tissue were established and named EEF, LYCB,LYCL and LYCMS respectively.DMEM/F12 culture medium, supplemented with beta- mercaptoethanol(2- ME),hepatocyte growth factor(HGF) and basic fibroblast growth factor(FGF-basic, human / murine),epidermal growth factor(EGF), Carboxymethylcellulose sodium(CMC), N- acetyl glucosamine(N-AG), was used as conditioned medium in our studies. European eel caudal fin blocks were treated by trypsin digestion to disaggregate tissues which were then transferred to flasks and placed reversely for 24h’s to attach disaggregated tissue on the surface of the culture flask. The conditioned medium was then added to flask to culture the fin primary cell. Through this method the primary cell and its cell line(EEF) were established successfully. The results showed that this conditioned medium is suitable for freshwater fish cells in primary culture and subculture.Based on above successful method, the conditioned medium was used for primary culture of large yellow croaker cell lines. Tissue blocks(brain, liver, muscle) of juvenile of large yellow croaker were used for culture without trypsin digestion. Three cell lines of large yellow croaker(LYCB, LYCL and LYCMS) were developed successfully. This result was also confirmed that the conditioned medium could be used for primary culture and subculture in vitro in both freshwater and marine fishes to meet the demand for nutrients, pH, and osmolality and so on.The proliferation curves of above established four cell lines in different passage proportions were detected. The impacts of growth factors on proliferation were analyzed by MTT assay. Theresults showed that different passage proportions and growth factors have an effect on the proliferation of the four cell lines. But, there is different effect in different cell line.Karyotypes of these 4 cell lines were also analyzed. The result showed that 54% of the EEF cell line’s metaphase cells have the chromosome number of 38. The proportion of LYCB, LYCL and LYCMS’s metaphase cells with chromosome number of 48 are 51%, 56% and 62%respectively. Relatively low proportion of the diploid number of chromosomes of the 4 fish cell lines suggested that chromosome number of these cell lines has been changed during culture in vitro. These differences of diploid number’s proportion between cultured cell lines and fish somatic cells in vivo are consistent with recognized knowledge. Furthermore, the transfection,cryopreservation and recovery of these cell lines were study primarily. The result showed that these four cell lines can be cryopreserved and recovered as well as used for the study of the expression of the exogenous gene too.