Research On Screening Of Serum-free Medium For Vero Cells And Cultivation Of H5N1 Influenza Virus

Influenza viruses which belong to the Orthomyxoviridae family influenza virus genus, is one of the major pathogens to threat human health. And vaccination is the most effective way to prevent the occurrence and spread. Traditional influenza vaccines are made by using chick embryo, which exists defects like contamination with exogenous agents, long period of the chick embryo supplement. Especially when the bird flu outbreak, resulting in massive death of chickens. It is difficult to meet the needs of large-scale production. African green monkey kidney cells linage (Vero) is one of the human vaccine production substrate recommended by WHO. Some researches have proved influenza virus could adapt to Vero cells. However, conventional Vero cell culture medium contains serum with complex ingredients. The residual serum could increase the difficulty for virus purification. In the meantime, when using Vero cell for influenza virus propagation, exogenous trypsin needs to be added for the lack of HAO lyase while serum could inhibit the activity of trypsin and further influence the yield of cell cultured influenza virus. These defects can be avoided by performing serum-free Vero cell cultured influenza virus. Vero cell serum-free medium development which will realize high efficient influenza vaccine production is an important segment in cell influenza vaccine research and development.The present commercial serum-free mediums development is aimed at the specific cell strain which have the specific serum-free medium ingredients. This study performed gradually decrease serum content for serum-dependent Vero cell adapting to serum-free medium. The adapted serum-free Vero cell (Serum Free Medium-Vero, SFM-Vero) cultured in different serum-free mediums including self-made low animal-based protein serum-free medium (Serum Free Medium-1, SFM-1), self-made non-animal-based protein serum-free medium (Serum Free Medium-2, SFM-2) and commercial serum-free mediums (VP-SFM、OptiPROTM SFM、EX-CELLTM VERO、VirusProTM VERO). The outcome indicated three serum-free mediums VP-SFM, SFM-1 and SFM-2, screened by cell shape and cell proliferation rate, could sustain SFM-Vero cell to proliferate and passage stably. The average glucose consumption of SFM-Vero cell in screened SFM-1 medium at 96 h was 11.306 mM, which was close to that in the normal medium contains serum. It indicates the substitution of serum in serum-free medium was able to stimulate cell to utilize glucose. Meanwhile, the average accumulated ammonia concentration in culturing was 4.8 mM, within a rational range with no inhibition cell growth and metabolism.In SFM-Vero cell culturing H5N1 influenza virus study, Box-Behnken design method was used to optimize pH, MOI and TPCK-Trypsin concentration. The result showed the factors’ response surface were all saddle-shaped, which indicates the response value exists maximum value. Based on the response surface and contour trend, the virus culture condition was further determined as:pH 7.64, MOI 0.01 and TPCK-Trypsin concentration 0.68 μg/mL. Under this condition, the HA titer of cultured influenza virus can be as high as 768 HAU/50μL, which is 1.5 times to H5N1 virus cultured in normal serum dependent Vero cell. Besides, the study tested some factors significantly affect H5N1 influenza virus yield in SFM-Vero cell including cell age, cultivation temperature and additional TPCK-trypsin. The result shows the hemagglutinin and TCID50 reached the highest level when H5N1 influenza virus inoculated in 36 h of SFM-cell age, with the average value 512 HAU/50 μL and 7.708 lgTCIDso/mL. The highest virus yield was achieved at temperature of 33 ℃, so was the corresponding HA value and TCID50, the average was 576 HAU/50 μL and 7.833 lgTCID50/mL. In the meantime, it is important to virus yield whether the initial concentration of TPCK-Trypsin should be replenished after 24 h in culturing virus process. In the course of H5N1 virus serial passage in SFM-Vero cell, the hemagglutinin titer and TCID50 of virus remained stable with the average value 435.2 HAU/50 μL and 7.85 lgTCID50/mL. Finally, HA and NA segment of A/Anhui/1/2005(H5N1) va virus by 5, 10,15 consecutive passages were sequenced. The result indicated there was no mutations of the two surface antigen gene of SFM H5N1.