Optipro

Vero-E6 cells of WHO origin (ECACC no 88020401) were obtained from Nuvonis (Vienna, Austria) and cultured according to the provided instructions. Briefly, 7.5 × 10⁶ cells were thawed, resuspended in 12 mL equilibrated OptiPRO™ (ThermoFisher Scientific, Waltham, MA, USA), supplemented with 4 mM GlutaMax (ThermoFisher Scientific, Waltham, MA, USA) and added to a T175 flask containing 88 mL of supplemented OptiPRO™. After 24 h incubation at 37 °C at 5% CO₂ and 95% relative humidity, media was replaced with 50 mL equilibrated supplemented OptiPRO™. The passaging cells were washed once with 13 mL of PBS before adding 5 mL of TrypLE (ThermoFisher Scientific, Waltham, MA, USA) to detach cells. Detached cells were resuspended in 15 mL OptiPRO™ and seeded at 40,000 cells/cm² for a 3-day passage. After two additional passages, cells were frozen at 4.5 × 10⁶ cells/vial in 1 mL of OptiPRO™ containing 7.5% DMSO in a Mr.Frosty freezing container (ThermoFisher Scientific, Waltham, MA, USA) at −80 °C and stored until further use.

To assess the toxicity of the shRNA expression using this system, we injected the pTol-GI-PB1-NP-NS vector into dorsal aorta of Hamburger and Hamilton (HH) stage 14–15 chicken embryos as previously described [23 (link)]. Briefly, 0.6 µg of pTol-GI-PB1-NP-NS vector and 1.2 µg of pTrans vectors were mixed with 45 µL of OptiPRO (Invitrogen) and incubated at room temperature for 5 min. In another tube, 3 µL of L2000 CD (Invitrogen) was added to 45 µL of OptiPRO and incubated for 5 min. These two solutions were mixed and incubated at room temperature for 20 min to allow complex formation, prior to injecting into embryos. Approximately 1–2 µL of complex was injected into the dorsal aorta HH stage 14–15 chicken embryo. To examine eGFP fluorescence, gonads from HH stage 40 recipient embryos were dissected and viewed under a fluorescence microscope for eGFP expression.

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.