Establishment and Validation of a Serum-Free Suspension Culture Process Using MDBK Cells for Efficient Production of OPIV3

Background The Madin-Darby Bovine Kidney (MDBK) cell line is a critical substrate for producing vaccines against Ovine parainfluenza virus type 3 (OPIV3). However, its adherent nature imposes substantial constraints on large-scale vaccine manufacturing using conventional culture systems. This study aimed to develop a serum-free suspension culture system for MDBK cells to produce OPIV3 and to validate its virus production capacity in a bioreactor. Results After the adherent MDBK cells were adapted to suspension culture via progressive serum reduction and serial passaging, several commercial serum-free media were screened. Bofit serum-free medium was identified as optimal for supporting consistent and high-density growth of these adapted cells, which were designated as MDBK-SM. Starting from an initial seeding density of 1.0 × 10⁶ cells/mL, the MDBK-SM cultures achieved a peak density of 2.0 × 10⁷ cells/mL. To demonstrate safety throughout the scale-up process, the cells were serially passaged to a high passage level (P30) and then tested for contaminants. Tests for bacteria, mycoplasma, and adventitious viruses were all negative. Tumorigenicity assays confirmed the non-tumorigenicity of MDBK-SM cells. Infection experiments demonstrated stable, high susceptibility to OPIV3 across various passages. Conclusions This study further optimized key infection parameters—including the initial cell density, multiplicity of infection (MOI), and harvest time—to establish efficient virus production conditions. Using these optimized parameters, OPIV3 was successfully amplified in a 5 L bioreactor, achieving a titer compliant with inactivated vaccine production standards. This work establishes a foundation for the safe, scalable, and controllable manufacturing of OPIV3 vaccines.