Optipro

Vero cells were seeded at a density of 3_x10⁴ cells/cm² in 125 cm² tissue culture flasks, cultivated for 24h and infected with recombinant virus at multiplicity of infection (MOI) of 0.001 in Opti Pro supplemented with 20 mM L-glutamine, 5 μg/ml trypsin and 0.5 μg/ml amphotericin B (all Sigma) in a total volume of 5 ml at 37°C for 1.5h. Culture medium was added to a final volume of 25 ml and infected cells cultivated for 48h observing cell lysis after 24 h. Virus titers were determined for Vero cells in virus growth medium containing Opti-Pro, 20 mM L-glutamine, 5 μg/ml trypsin and 0.5 μg/ml amphotericin B (Sigma) by endpoint dilution assay. Vero cells were seeded at a density of 1.5_x10⁴ cells/well the day before infection, viruses were diluted from 10⁻³ to 10⁻⁹ in virus growth medium, then cells were infected. Three days post infection cells were analyzed for cytopathic effects by confocal light microscopy. Virus titers were calculated as 50% tissue culture infectious dose per ml (TCID₅₀/ml) according to the Reed and Muench method [39 (link)].

An egg-derived influenza H7N9 reassortant vaccine virus (NIBRG-268) was generated using reverse genetics by the UK National Institute of Biological Standard and Control (NIBSC) and supplied to National Health Research Institutes (NHRI), Taiwan. The NIBRG-268 virus contains six internal genes of the egg-adapted high-growth A/PR8 virus and two surface protein genes (HA and NA) of A/Anhui/1/2013(H7N9). At NHRI, the NIBRG-268 virus could not grow well initially in Madin-Darby canine kidney (MDCK) cells (∼10⁵ TCID₅₀/ml). Therefore, we serially passed the NIBRG-268 virus in MDCK cells to increase its growth efficiency. MDCK cells (ATCC CCL-34) were purchased from the Food Industry Research and Development Institute, Hsinchu, Taiwan. MDCK cells were grown using DMEM (GibcoBRL) plus 5% fetal bovine serum (Moregate) to generate master and working cell banks following cGMP guidelines and have been characterized to fulfill the requirements for continuous cell lines used for manufacture of biological products [12 (link)–14 (link)]. For vaccine production, serum-free medium (OptiPro, Life Technologies) was used for cell growth and OptiPro supplemented with 2 μg/ml of TPCK-trypsin (Sigma) was used for virus replication.