# Suspension cultivation of mosquito cell lines for the production of the mosquito-borne flavivirus Usutu virus in a stirred-tank bioreactor

## TL;DR

Researchers developed a serum-free method to grow mosquito cells in bioreactors, enabling efficient production of mosquito-borne viruses like Usutu virus.

## Contribution

First demonstration of serum-free suspension cultivation of C6/36 mosquito cells in a bioreactor for virus production.

## Key findings

- C6/36 and U4.4 mosquito cell lines achieved high concentrations in serum-free suspension conditions.
- C6/36 cells in a bioreactor produced Usutu virus at high titers with efficient growth kinetics.
- This system offers a scalable approach for producing mosquito-borne viruses and biotech products.

## Abstract

Mosquito-borne pathogens are a global health burden. The realization of a scalable serum-free mosquito cell suspension cultivation method would enable the production of novel biotechnological products, including vaccines, recombinant proteins, and (modified) viruses for biological control. This study reports the adaptation of two Aedes-derived cell lines, C6/36 and U4.4, to serum-free suspension conditions using the chemically defined EX-CELL medium. Both cell lines achieved high cell concentrations (> 1.0 × 107 cells/mL) in shake flasks with minimal adaptation. The C6/36 suspension cultures were subsequently transferred to a 500 mL stirred tank bioreactor (STR), which likewise achieved high cell concentrations (1.3 × 107 cells/mL) and a doubling time of 31–36 h. As a proof of concept for the production of a virus in this system, C6/36 cells were cultivated in a STR and infected with the mosquito-borne flavivirus Usutu virus (USUV). These cultures produced high virus titers (> 3.6 × 108 TCID50/mL), with similar growth kinetics compared to adherent and shake flask cultures. This work is the first to show C6/36 mosquito cell cultivation in a bioreactor and demonstrates the potential of this serum-free suspension culture system for the efficient production of mosquito-infecting viruses.

The online version contains supplementary material available at 10.1038/s41598-025-33792-z.