Laboratory-Scale Cultivation of the Ospa-Vaktsina Vaccinia Virus Strain in Vero Cells on Bionoc Ii® Macrocarriers Using the Bellostage™-3000 System

Vaccinia virus remains relevant as a viral substrate for smallpox vaccines and as an orthopoxvirus model. This study evaluated, at laboratory scale, the feasibility of cultivating the “Ospa-vaktsina” vaccinia virus strain in Vero cells grown on BioNOC II® macrocarriers using the BelloStage™-3000 Tide Motion system. The study was not designed as a comparative assessment of BelloStage™-3000 performance against another cultivation platform. Vero cells were cultivated in BelloCell®-500A vessels in Dulbecco’s Modified Eagle Medium supplemented with 2% fetal bovine serum. After 120 h of cell cultivation, when the total cell number reached 1.99 × 10⁹ cells per vessel, cultures were infected with a working virus inoculum at a calculated infectious input of 0.01 TCID₅₀ per cell. The infectious input was calculated as the total TCID₅₀ delivered in the 500-mL inoculum divided by the total cell number at infection. One bioreactor run included three parallel BelloCell®-500A vessels (n = 3 culture units within a single run). Cell attachment to the macrocarriers was 89% at 3 h after seeding. The total cell number increased from 7.45 × 10⁷ cells at seeding to 1.99 × 10⁹ cells per vessel at 120 h, corresponding to a 26.7-fold increase. Following infection, the virus titer increased from 4.60 log₁₀ TCID₅₀/mL in the working inoculum to a maximum of 6.00 log₁₀ TCID₅₀/mL at 120 h post-infection, a 1.40-log₁₀ (approximately 25.1-fold) increase. Culture medium parameters were maintained within predefined operating ranges for the cell-growth and post-infection phases. Under the conditions tested, the Vero/BioNOC II® culture supported replication of the “Ospa-vaktsina” strain in BelloStage™-3000. Because the study comprised a single bioreactor run and did not include a parallel comparator platform, the findings demonstrate laboratory-scale feasibility but do not establish technological superiority, process scalability, or suitability for vaccine manufacturing.