Abstract: The objectives of this study were to screen a serum-free medium suitable for suspension culture of BHK21 cells in bioreactor, and to achieve pseudorabies virus proliferation by using this this technique. BHK21 cells were serum-freely acclimated by screening the commercially available serum-free medium using cell morphology, viability and multiplication ratio as evaluating indicators. The parameters of serum-free suspension culture of BHK21 cells were determined in 10 L and 40 L bioreactors. Furthermore, a pseudorabies virus proliferation test was carried out by using the screened serum-free culture medium and suspending cultured BHK21 cells. The results showed that the commercially available mediumⅠwas consistent with the screening requirements, and the cultured cells were uniform in morphology and less in clusters. The proliferation ratio of the cells cultured for 2 days was 3.5 times, and the cell viability was above 95%. The optimum parameters of suspension culture of BHK21 cells in bioreactor were as follows the rotation speed was 80 r/min, pH value was 7.2, and DO was 60%. Under this condition, when the inoculated BHK21 cell density was 6×10⁶ cells/mL and the inoculated pseudorabies virus dose was 0.3%, the titer of the harvested viruses at 48 h reached up to 10^9.5 TCID₅₀/mL. The serum-free suspension proliferation technique of pseudorabies virus was achieved in this study, providing the possibility of optimizing its production process.

Key words: bioreactor, serum-free medium, parameter, pseudorabies virus