Establishment of a Suspension MDBK Cell Line in Serum-Free Medium for Production of Bovine Alphaherpesvirus-1.

Pengpeng Wang,Xinrui Tian,Shulin Huang,Chen Peng,Zhanhui Wang,Letu Ge,Haoran Zhao,Chengwu Hao,Mengyao Liu,Wenxue Wu

doi:10.3390/vaccines9091006

Abstract

The Madin–Darby bovine kidney (MDBK) cell line is currently used for the production of bovine alphaherpesvirus-1 (BoHV-1) vaccine. For the purpose of vaccine manufacturing, suspension cells are preferred over adherent ones due to simplified sub-cultivation and an easier scale-up process, both of which could significantly reduce production cost. This study aimed to establish a procedure for the culture of BoHV-1 in the suspended MDBK cell line in serum-free medium. We screened several commercially available serum-free media and chose ST503 for subsequent experiments. We successfully adapted the adherent MDBK cells to suspended growth in ST503 in the absence of serum. The maximum density of suspension-adapted MDBK cells could reach 2.5 × 107 cells/mL in ST503 medium with optimal conditions. The average size of suspension-adapted cells increased to 18 ± 1 µm from 16 ± 1 µm. Moreover, we examined tumorigenicity of the suspended cells and found no sign of tumorigenicity post adaptation. Next, we developed a protocol for the culture of BoHV-1 in the cell line described above and found that ultrasonic treatment could facilitate virus release and enhance virus yield by 11-fold, with the virus titer reaching 8.0 ± 0.2 log10TCID50/mL. Most importantly, the prototype inactivated BoHV-1 vaccine we generated using the suspension cultures of MDBK cells induced neutralizing antibodies to a titer comparable to that of the commercial inactivated BoHV-1 vaccine. Overall, we established and optimized a protocol for the production of inactivated BoHV-1 vaccine in MDBK cells adapted for suspension culture, which provides insights for future large-scale manufacturing of BoHV-1 vaccine.

Highlights

Adaptation of adherent cells to serum-free suspension culture is a procedure widely used in the production of biological products such as vaccines, monoclonal antibodies and pharmaceutical protein products [1,2,3]
Studies have shown that production of bovine adenoviruses (BAdV) in suspended Madin–Darby bovine kidney (MDBK) cells led to higher viral yields than that in adherent cells [12]
The virus productivity increased to 8.0 ± 0.2 log10TCID50/mL in the suspension culture when the cell density at infection increased to 8.0 × 106 cells/mL from 0.9 × 106 cells/mL. These results indicate that the virus productivity of suspension MDBK cells was lower than that of adherent MDBK cells, we could increase the production of bovine alphaherpesvirus-1 (BoHV-1) by increasing the initial cell density, which is only possible with suspended MDBK cells

Summary

Introduction

Adaptation of adherent cells to serum-free suspension culture is a procedure widely used in the production of biological products such as vaccines, monoclonal antibodies and pharmaceutical protein products [1,2,3]. Compared to adherent tissue culture, serum-free culture can reduce the cost and improve the quality and stability of biological products. MDBK cells are permissive to a variety of viruses and are usually adherently cultured in roller bottles or on microcarriers for mass production of vaccines in industry [10,11]. Compared to adherent tissue culture, suspension culture is more space efficient and can support the growth of cells with higher concentration, which is more suitable for industrial production. Studies have shown that production of bovine adenoviruses (BAdV) in suspended MDBK cells led to higher viral yields than that in adherent cells [12]

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