Limited subculture and biological characteristic analysis of three cell lines from fat greenling(Hexagrammos otakii)

Fat greenling(Hexagrammos otakii)is one of the important commercial fishes along the northern coast of China,and its artificial breeding and culture have been developed recently.Primary culture of fin,lip and kidney from H.otakii was studied using tissue explant method.Until now cells of three tissues have been subcultured at passage 30 for fin,passage 31 for lip,passage 35 for kidney respectively.The results showed that the fin and lip tissues could be dispersed quickly after being digested with 0.5% hyaluronidase and 0.2% collagenase Ⅱ.The optimal growth conditions for fin,lip and kidney tissues were 20% fetal bovine serum(FBS),5 ng/mL basic fibroblast growth factor(bFGF),20 μg/mL chondroitin sulfate and 40 ng/mL insulin-like growth factor-I to medium DMEM/F12(pH 7.2)at 25 ℃.The doubling time of fin,lip and kindey cell numbers was about 58.7 h,50.4 h and 32.9 h at the 20th passage.Karyotype analysis of 100 metaphase plates revealed that the feature diploid chromosome number was 2n=48 in fin,lip and kidney cell at 25th passage.The cell livability of these three kinds of cell were(84.59±1.07)%,(85.75±1.03)% and(87.39±1.05)%,respectively,when recovered after being stored in liquid nitrogen for 60 d at the 20th.Now,the three kinds of cell have been preserved in China Center for Type Culture Collection(CCTCC).The methods of cell culture in vitro could be used to prevent and cure fish diseases and the pathomechanism study.