Microbial, Cellular and Molecular Biology

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Browsing Microbial, Cellular and Molecular Biology by Subject "Adaptation"

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    Sequential Adaptation of Vero Cell Lines in Serum Free Medium for Fixed Rabies Virus Propagation
    (Addis Ababa University, 2020-03-03) Mohammed, Jemal; Desalegn, Asnake (PhD)
    Culturing cell is a process of growing cells under physically controlled and aseptic environment in artificial medium. There are several cell lines including vero cell lines propagated using this method. Vero cell lines are derived from kidney of the African green monkey (Cercopithecus aethiops). They are continuous and anchorage based cells that need sufficient surface to proliferate in growth medium. Vero cells are most commonly used to detect bacterial toxins and production of different vaccines. It has also used to grow different strains of rabies viruses to produce cell culture based vaccine for human and animal use; however vero cells propagated in serum supplemented medium that is very expensive and source of different contaminant agents. The aim of this research was therefore to adapt Vero cell lines in serum free medium sequentially for propagation of Pasteur virus (PV) and Evelyn Rokitnicki Abelseth (ERA) rabies virus strains. Vero cells were adapted sequentially in serum supplemented media by gradually reducing from 10% to 0% of serum concentration. Viable cells were counted until passage seven in each serum concentrations. The maximum viable cell density of Vero cells at each serum supplemented medium (0%, 1%, 2.5%, 5%, 7.5% and 10%) was 2.86x106 cells/ml, 2.75x106 cells/ml, 2.75x106 cells/ml, 2.70x106 cells/ml, 2.92x106 cells/ml and 2.75x106 cells/ml, respectively. The preferable incubation times to obtain those maximum viable cells were 96 hours and 144 hours for vero cells proliferated in 2.5% - 10% and 0% - 1% supplemented medium respectively. The incubation time increased as serum concentration in growth medium decreased to reach the confluence stage. The yield of viable cells grown in serum free medium were similar to cells proliferated in a serum supplemented medium. The virus titration was carried out at each serum concentration proliferated cells to determine the virus titer of both viruses. The maximum recorded virus titers for PV was 105.36TCID50/ml with 0.01 Multiplicity of infection after 96 hours incubation on serum free grown cells propagated virus and the maximum virus titer for ERA was 105.61TCID50/ml with 0.001 Multiplicity of infection on serum free media propagated viruses after 120 hours incubation. The incubation times were also increased as serum concentration was decreased to obtain the higher titer in both virus strains. From the results of this study, it can be concluded that cells that were adapted to serum free conditions is suitable for use in the rabies vaccine production.