Clin Exp Vaccine Res.  2020 Jan;9(1):48-55. 10.7774/cevr.2020.9.1.48.

Suspension culture of Vero cells for the production of adenovirus type 5

• Lee DK
• Park J
• Seo DW

• KMID: 2470837
• DOI: http://doi.org/10.7774/cevr.2020.9.1.48

Abstract

PURPOSE
Most cell culture processes for viral vaccine production are mainly based on adherent cell culture systems using serum, which are associated with expensive and labor-intensive processes to produce large amounts of viral vaccine strains. In this study, we investigated whether Vero cells could be grown in serum-free and shaking suspension conditions. Furthermore, we assessed the ability of the Vero cell suspension culture system to produce adenovirus type 5 (Ad5), compared to that of the adhesive Vero cell culture system.
MATERIALS AND METHODS
We tested the feasibility of commercial serum-free media for Vero cell culture. For the adaptation of Vero cells in suspension culture, adhesive Vero cells were added in the early phase of shaking suspension culture, and 50 days after shaking suspension culture, suspension-adapted Vero cells were subcultured continuously. To assess the virus production ability of Vero cells in suspension, the cells were infected with Ad5-green fluorescent protein and evaluated based on their fluorescence intensity.
RESULTS
The Vero cells grown in OptiPRO serum-free medium showed no changes in morphology and growth rate, but MRC-5 and FRhk-4 cells showed morphological changes and decreased growth rate, respectively. The Vero cells were well adapted to the suspension culture system. The Vero cells in suspension showed a better Ad5 production ability than the adherent Vero cells.
CONCLUSION
Vero cells can be grown in OptiPRO serum-free medium. Further, our suspension culture-adapted Vero cells may be suitable to produce viral vaccine strains due to their high ability to produce viruses such as Ad5.

Keyword

Vero cells; Serum free culture; Suspension culture; Adenovirus type 5

MeSH Terms

Adenoviridae*
Adhesives
Cell Culture Techniques
Culture Media, Serum-Free
Fluorescence
Vero Cells*
Adhesives
Culture Media, Serum-Free

Figure

• 

  Fig. 1 (A) Morphology of Vero cells grown under serum-free conditions. The blue and red arrows represent abnormal cell growth. (B) Growth rate of cells cultured in serum-free conditions, compared with that of cells cultured in DMEM containing 10% FBS (control; blue line). DMEM, Dulbecco's Modified Eagles Medium; FBS, fetal bovine serum.

• 

  Fig. 2 (A) Effect of serum-free culture on cell morphology. (B) Proliferation rate of MRC-5 cells in serum-free media compared with that in DMEM containing 10% FBS (blue line). (C) Morphology of FRhk-4 cells in serum-free culture conditions. (D) Growth rate of FRhk-4 in serum-free culture conditions compared with that in DMEM containing 10% FBS. DMEM, Dulbecco's Modified Eagles Medium; FBS, fetal bovine serum.

• 

  Fig. 3 (A) Adaptation of Vero cells to suspension culture conditions. Adhesive Vero cells (1.6×105/mL) were cultured in a shaking flask and their viability was measured. The cell viability was monitored every 5 days by trypan blue staining. The starting cell concentration of subculture was adjusted using adhesive Vero cells (1.6×105/mL). (B) The morphology of Vero cells cultured under adhesive and suspension culture conditions after Ad5 virus infection at an multiplicity of infection of 0.1. (C) After infection, fluorescence intensity of green fluorescent protein in both cell types was measured by using a fluorescence plate reader. Ad5, adenovirus type 5.

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