Nunc easyfill cell factory system

The rVSV-S virus was replicated in Vero cells (WHO Vero RCB 10–87) and widely accepted by the WHO and regulatory agencies for the manufacturing of human viral vaccines [57 (link)] using serum-free medium (SFM) (FLEX20, Biological Industries, Beit Haemek Israel). Vero cells were seeded into 6342 cm² multi-trays (MTs) (NUNC EasyFill Cell Factory System, Thermo Fisher Scientific, MA, USA cat. # 140400) at an initial density of 1,500,001 cells/cm² and a total volume of 1000 mL SFM FLEX20 medium. Typically, 3 to 6 MTs were grown in parallel, resulting in a working volume of 3 to 6 L. Cells were grown to confluence in MT at 37 °C and 5% CO₂ and then infected with an estimated 0.1 multiplicities of infection (MOI) of rVSV-S. The inoculated cells were maintained for 72 h in MT at 37 °C and 5% CO₂ and harvested into 3–6 one-liter flasks.

The rVSV-∆G-spike vaccine was produced in BioBLU^® 5p Eppendorf single-use bioreactors (Hamburg, Germany) [6 ] or in multi trays (MT) (NUNC EasyFill Cell Factory System, Thermo Fisher Scientific, Waltham, MA, USA). Vero cells were absorbed to fibra-cel within the bioreactor/MT and grown for six days. Cells were then infected with the vaccine virus, which was allowed to replicate in the cells for 48 h. Infection was carried out at a multiplicity of infection (MOI) of 0.1 (The MOI refers to the number of virions that are added per cell during an infection; an MOI of 0.1 means that 1 virion is added per 10 cells). The medium from the bioreactor/MT, containing the released vaccine virus, was harvested into a Flexboy bag (Sartorius, Gottingen, Germany). The bag was transferred to the downstream process.