# Efficient Serum-Free Rabies Virus Propagation Using BSR and Vero Cell Lines: A Comparative Evaluation of BioNOC II® Macrocarriers in the BelloStage™-3000 Bioreactor Versus Conventional Microcarriers

## TL;DR

This study compares new methods for growing rabies virus in cells, showing that a bioreactor with special carriers produces more virus faster and safer than traditional methods.

## Contribution

The study introduces a novel bioreactor and macrocarrier system for serum-free rabies virus propagation, achieving higher yields and biosafety.

## Key findings

- Using BioNOC II® macrocarriers and the BelloStage™-3000 bioreactor significantly increased rabies virus titers in BSR and Vero cells.
- Viral titers in BSR cells reached 5.6 × 108 FFU/mL, surpassing microcarrier systems by 19.3-fold and 15.3-fold.
- The system supports dense cell layers and reduces contamination risks due to serum-free medium use.

## Abstract

Rabies is a deadly disease that can infect both animals and humans. One of the most reliable ways to prevent it is through the vaccination of animals. This study demonstrates an effective approach to culturing rabies virus using BSR and Vero cell lines. The cells were cultured in the serum-free nutrient medium OptiPRO™ SFM, which reduces the risk of microbial contamination. Two types of carriers were used for cell growth: BioNOC II® macrocarriers and Cytodex 1 and Cytodex 3 microcarriers. Cultivation on macrocarriers was carried out in the BelloStage™-3000 bioreactor, which operates on the principle of “Tide motion”, ensuring gentle mixing and protecting the cells from damage. Cultivation on microcarriers was performed in flasks with a magnetic stirrer. The results showed that the use of macrocarriers in combination with the bioreactor system BelloStage™-3000 made it possible to obtain significantly more viral material in a shorter time. This approach makes vaccine production faster, safer, and more efficient. This is especially important for regions where rabies still poses a serious threat to the health of animals and humans.

The rabies virus remains a significant public health threat, particularly in regions with limited access to vaccination. This study shows that the BelloStage™-3000 bioreactor, operating on the “Tide Motion” principle, in combination with BioNOC® II macrocarriers, ensures highly efficient rabies virus cultivation in BSR and Vero cells grown in serum-free OptiPRO™ SFM medium. This system supports effective cell attachment, formation of a dense and metabolically active cell layer, and reduces microbial contamination risks associated with serum-containing media. For comparison, rabies virus cultivation was also performed on Cytodex 1 and Cytodex 3 microcarriers in spinner flasks. The use of the BelloStage™-3000 bioreactor system with BelloCell™ 500A disposable vials and BioNOC II® macrocarriers resulted in significantly higher virus titers compared to traditional Cytodex 1 and Cytodex 3 microcarrier culture systems. Thus, in the BSR cell culture, the maximum virus titer reached 5.6 × 108 FFU/mL by day 4 of cultivation, which exceeded the titers obtained on Cytodex 1 and Cytodex 3 microcarriers by about 19.3-fold and 15.3-fold, respectively. A similar trend was observed for the Vero cell line: the peak titer was 2.0 × 108 FFU/mL by day 5 of culturing, which was higher than the values obtained on Cytodex 1 and Cytodex 3 by about 14.0-fold and 9.6-fold, respectively. These findings demonstrate that the integrated use of BioNOC® II macrocarriers, the BelloStage™-3000 bioreactor, and a serum-free medium provides a scalable, reproducible, and biosafe platform for rabies virus production, offering substantial advantages over traditional microcarrier-based systems.