# Engineering AQP1-Deficient DF-1 Suspension Cells for High-Yield IBDV Production and Vaccine Scale-Up

## TL;DR

This paper shows that modifying DF-1 cells to grow in suspension improves the production of a poultry virus vaccine, making large-scale manufacturing more efficient.

## Contribution

AQP1 disruption enables DF-1 cells to grow in suspension, enhancing IBDV production for scalable vaccine manufacturing.

## Key findings

- AQP1-deficient DF-1 cells grew as suspension cultures with high viability and density.
- Suspension DF-1/AQP1− cells produced significantly higher IBDV titers than monolayer or wild-type cells.
- Virus production time was reduced in suspension cultures compared to traditional methods.

## Abstract

Background: Large-scale production of poultry viral vaccines increasingly requires robust suspension cell platforms. However, most avian cell lines, including DF-1, are strictly anchorage-dependent, limiting scalability. Aquaporin-1 (AQP1) regulates cell–cell adhesion and membrane dynamics, making it a potential target for engineering suspension growth. This study aimed to generate a stable DF-1 suspension cell line via AQP1 disruption and evaluate its potential for enhanced infectious bursal disease virus (IBDV) production. Methodology: DF-1 cells were engineered using a CRISPR/Cas9 ribonucleoprotein system to create a truncated AQP1 gene. DF-1/AQP1− cells were assessed for morphology, tumorigenicity in nude mice, and genetic stability across 20 passages. Suspension growth, cell density, and viability were measured. Cells were infected with IBDV strain BJQ902, and viral titers were compared with wild-type DF-1 and monolayer DF-1/AQP1− cells. Results: DF-1/AQP1− cells maintained normal morphology, were non-tumorigenic, and retained stable AQP1 mutations. They grew as true suspension cultures without adaptation, reaching 4.0 × 106 cells/mL with >95% viability. Suspension DF-1/AQP1− cells cells produced significantly higher viral titers (9.0 log TCID50/mL; 8.63 log EID50/mL) than both monolayer DF-1/AQP1− and wild-type DF-1 cells. Virus production time was shortened in suspension cultures. Conclusions: Targeted AQP1 disruption converts DF-1 cells into a stable, non-tumorigenic suspension cell line with markedly enhanced IBDV production, providing a scalable platform for next-generation avian vaccine manufacturing.