Establishment Of Highly Sensitive MDCK Monoclonal Cell Line With Influenza A H1N1 Influenza Virus

The H1N1 influenza virus is an extremely important member of the influenza A virus family.It is the main pathogen of the global influenza outbreak and poses a great threat to human health.Daily general prevention and antiviral drugs are not effective in preventing infections in humans.Currently,the most effective way to prevent the H1N1 influenza virus is to vaccination the flu vaccine.The traditional production process of influenza vaccines uses chicken embryos as a production substrate and has many disadvantages.WHO recommends the use of MDCK cells as an excellent production substrate for influenza vaccines.Large-scale cultivation of cell bioreactors to produce viral vaccines is the only way for the development of biomedical industry in China,and finding the ideal cell as the production base of influenza vaccine is one of the key steps in this process.In this paper,a MDCK monoclonal cell bank was prepared and established by limiting dilution method.Two high-sensitivity adaptive MDCK monoclonal cell lines of influenza A H1N1 influenza virus were obtained by screening,and the growth characteristics and proliferation of the two cells were obtained.In the study,cells were assayed according to the requirements of the Pharmacopoeia of the People's Republic of China.The main obtained findings were as follows:1.Introduced an adherent cultured MDCK cells from CCTCC and prepared MDCK monoclonal cells by limiting dilution method.By exploring the effects of serum type,serum concentration and single-cell density on the preparation of monoclonal cells,the ideal limiting dilution method was used to prepare were fetal bovine serum concentration of 15%and the single-pore inoculation density of 1 per well.A total of 114 MDCK monoclonal cells were obtained,providing a basis for cell resources for subsequent experiments.2.Resuscitated MDCK monoclonal cells,the average cell viability was 0.954±0.016.92.1%of the cells growing up time were over 3 days;66 cells were growth with good status,accounting for 57.9%;MDCK monoclonal cells have three cell morphology,including polygonal epithelial,fibroblast-like and island-like.Two high-sensitivity adaptive cell lines of influenza A?H1N1?virus,MDCKCL118 and MDCK CL134,were screened by measuring the hemagglutination titer value.The blood coagulation titer value reached 1:128,and the MDCK monoclonal cell working library was 1:32.The TCID₅₀0 values of MDCK CL118,MDCK CL134 and MDCK working bank cells were7.23log10TCID₅₀/mL,7.4log10TCID₅₀/mL,6.33 log10TCID₅₀/mL,respectively.MDCK monoclonal cells maintained a higher and stable hemagglutination titer after multiple passages.3.Established a master cell bank and a worked cell bank,and performed verification according to the protocol.The morphology of monoclonal cell lines MDCK CL118 and MDCK CL134 was consistent with the official website.The recovery rates of the monoclonal cell lines MDCK CL118 and MDCK CL134 were 95.44%and 94.95%,respectively,and the densities were 22.3×10⁴/mL and 23.5×10⁴/mL,respectively.The maximum value-added concentrations were 66.6×10⁴/mL and 38.4×10⁴/mL,respectively.The doubling time was 18.7h and 23.17h,respectively,and the growth curves were all“S”.The number of chromosomes 2n=74-76 was 30 and 31,respectively,accounting for 58%and 62%,respectively.Lactate dehydrogenase isoenzyme experiments,the number of bands of MDCK CL118,MDCK CL134,MDCK mother cells were three,the migration distance of the three cells was completely the same,and the two cells and MDCK mother cells were of the same species.By sterility examination,mycoplasma examination,intracellular and exogenous viral factor test,MDCK CL118 and MDCK CL134 monoclonal cell lines were negative.