Stability Of Growth And Influenza Virus Production Of MDCK Suspension Cells During Long-term Subculture

Madin Darby Canine Kidney(MDCK)cells have been widely studied as one of the most commonly used cell line for influenza virus vaccine production.Previous studies have found that adherent MDCK cells,as a continuous cell line,suffer from instability problems such as changes in cell morphology and growth capacity and a decline in influenza virus production efficiency during long-term culture.However,the stability of influenza virus production using serum-free suspension MDCK cells during long-term culture is not clear yet.Therefore,it is great importance to investigate the change of morphology and growth of suspended MDCK cells during long-term subculture and the stability of influenza virus production.In this work a H9N2 avian influenza virus strain and a suspension MDCK cell line were used for investigation.First,the suspension MDCK cells were passaged over 200 days,and various passages of P1,P29,P63,P88,and P111 were obtained.The cells of 5 passages were found to show the same morphology,additionally with no obvious clumps.In terms of the stability of virus production,suspension MDCK cells of P1,P29,P88 and P111 were used.The results showed that both the virus titer(HA titer)and the specific virus yield(Svy)increased with the increase of cell passage number,and the maximum HA titer(HAmax)of P111 cells was 0.47 Log2HAU/100 ?l higher than that of P1 cells.Next,P1 cells and P111 cells were used to investigate the impacts of operating conditions regarding substrate suppling,MOI,pH and lactic acid in the infection phase on the stability of virus production.To study whether the unstable characteristics of influenza virus production in suspension.The results showed that under each experimental condition,HAmax and maximum specific virus yield(Svymax)of P111 were higher than those of P1 cells.The difference between HAmax was 0.15-1.09 Log2HAU/100 ?l,and the difference between Svymax was 3505.28-15779.11 virions/cell.The results confirmed that the high-yield characteristics of high-passage suspension MDCK cells are not affected by the operating conditions of the production process.P1 and P111 cells were used to investigate the characteristics of producting influenza virus with MDCK suspension cells of high and low passages.The results showed that in the early stage of infection phase,the release of influenza virus particles from P111 was faster than P1 cells with a maximum difference was 6161.88 virions/cell/h at 6-7 hpi.In addition,it was found that the proportion of G0/G1 phase cells in P111 cells was about 10%higher than that of P1 cells;the intracellular ROS level of P111 cells was lower than P1 cells.The levels of ROS in P111 cells at 0 hpi and 6 hpi were 47.00%and 83.11%of P1 cells,respectively.With the use of transcriptome methods,the gene expression levels of P1 and P111 cells were analyzed.It was found that the differential expressed genes were mainly enriched in MAPK,PI3K-Akt,endocytosis,and ABC transporter signaling pathways.Additionally,the protein genes related to promoting G0/G1 phase arrest and ROS had changed significantly.Overall,the stability of cell growth and influenza virus production during the long-term subculture of suspension MDCK cells were verified,and the reasons for the difference in the virus production efficiency of high-passage and low-passage cells were preliminarily investigated.The research results provide an important technical support for the development of the influenza virus production with suspension MDCK cells.