Parameters for MDBK cell growth on microcarriers and BoHV-1 virus production

Ethel C. Freitas, Otto D. Mozzer, Carla M. B. Freitas, Adelson G. Costa, Ronaldo Zucatelli Mendonça

Abstract


Bovine herpes virus 1 (BoHV-1) is an important veterinary agent , which causes infectious bovine rhinotra-cheitis. This disease affects the respiratory tract or genitals, causing weight loss, reduced milk production and abor-tion. Several vaccines against BoHV-1 have been developed. In this paper, we study the parameters for MDBK growth on microcarriers (Cytodex 1) and for BoHV-1 virus production. The cell culture attached to microcarriers is an effi-cient method to enlarge the surface of cell growth and for large-scale cell production. Our studies reveal that MDBK adhered to MCs in 30 minutes and that initial agitation of culture did not influence on the efficiency of adhesion or cell growth. In our experiments, we detected no relevant influence of agitation on initial cell adhesion of MDBK to MCs. The maximum cell yield was similar to all initial conditions of agitation studied. The maximum yield obtained in culture started with 15, 20 and 30 cells / MC, was respectively.8.7 x105, 9.3 x105 and 9.8 x105 cells / ml . The cellular distribution on the MCs at the beginning of the culture was more heterogeneous in higher initial densities. After three medium exchanges during MDBK cell culture, the increase in the final yield was 100% higher than that from culture performed without medium change (0.93 x 106 cells / mL). Replacing 50% of the culture medium with fresh medium after 24 hours of growth, the concentration of glucose (5 mM) and glutamine (1.8 mM) were almost completely res-tored. In these studies, BoHV-1 infections of MDBK were performed after 48, 72 and 86 hours with daily exchanges of 50% of the medium. The increase in viral titer was proportional to the number of viable cells present at the time of infection. The best result of BoHV-1 production was achieved when the infection was performed from 86 hours of cell culture, reaching about 3.7 x108 (TCID50/ml) after 24-48 hours of infection, being on average four times higher when compared to the culture in which the infection was performed with 48 hours of culture and approximately 2 times greater than the crop whose infection occurred after 72 hours of culture. The best yields are obtained when viral infections were performed on cultures with higher cell densities. The best result for the production of BoHV-1 occur-red with 10 MOI, 48 hours after infection, which yield was 24 and 41% superior to the MOI 0.1 and 1, respectively.

Keywords


MDCK cells; microcarriers, BoHV-1; bovine herpes virus

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DOI: http://dx.doi.org/10.17525/vrrjournal.v22i1.326

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