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

<b>Background</b>: 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. <b>Methodology</b>: 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. <b>Results</b>: 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. <b>Conclusions</b>: 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.