Cell Density Effects in Different Cell Culture Media and Their Impact on the Propagation of Foot-And-Mouth Disease Virus

Abstract

Foot-and-mouth disease virus (FMDV) is endemic in many parts of the world. Vaccination is an important control measure, limits viral spread, and can help to eradicate the disease. However, vaccination programs are cost-intensive because of the short shelf life of vaccines and the need for frequent re-vaccination. Animal-component-free (ACF) or chemically defined media (CDM) at high cell densities are a promising approach for the production of inexpensive high-quality vaccines, but the occurrence of cell density effects has been reported for various virus-cell systems in vaccine production. For FMDV, the use of CDM or ACF media for vaccine production has not been studied and no information about cell density effects is available. This work describes the propagation of FMDV in ACF or in CDM. Cells were grown at increasing cell densities and either 100% media exchange or addition of 30% fresh media was performed before infection with FMDV. Increasing cell densities reduced the viral titer and increased yield variability in all media except BHK300G. This effect can be mitigated by performing a 100% media exchange before infection or when using the controlled environment of a bioreactor. The media composition and also a fragile relationship between virus and cell metabolism seem to be causal for that phenomenon.

Keywords: animal-component-free media; antifoam; cell density; chemically defined media; foot-and-mouth disease virus; suspension cells.

Figure 1

Impact of antifoam in the cell culture media on viral yield in infected cultures and cell viability in the absence of infection. Antifoam was added to the media at a final concentration of 0.03, 0.3, 1.7 or 3.3% (v/v). A culture with no additives served as a control. (a) Cells at a density of 1 × 10⁶ cells/mL were infected with Foot-and-mouth disease virus (FMDV) O SAU/18/2018 at a multiplicity of infection (MOI) of 0.01 and the viral harvest at 20 hpi is shown as log₁₀ TCID₅₀/mL. (b) Antifoam was added to non-infected cells (1 × 10⁶ cells/mL) and cell viability was measured after 20 h of culturing. Experiments were performed three times independently. Significance code: “****” p ≤ 0.0001.

Figure 2

Cell density experiments in spin tubes and 3L-single-use bioreactors with different cell culture media for cell growth. Cells were grown in (a,d) the animal-component-free (ACF) media BHK200 or in the CDM (b,e) BHK300B and (c,f) BHK300G in three different cell densities (1 × 10⁶/2 × 10⁶/3 × 10⁶ cells/mL). A media exchange of either 100% or addition of 30% fresh media was performed before infection with FMDV A24 Cruzeiro (panels a–c) or O/SAU/18/2015 (panels d–f) at a MOI of 0.01. The final virus titers in log₁₀ TCID₅₀/mL of the spin tube experiments are colored in blue, while the results of the bioreactor runs are shaded in red. Significance code: “**” p ≤ 0.01.

Figure 3

Virus growth in cell culture media with different contents of conditioned media in spin tubes. Cells were grown in the (a) ACF media BHK200 and in the chemically defined media (CDM) (b) BHK300B and (c) BHK300G in two different cell densities (1 × 10⁶ cells/mL: blue; 3 × 10⁶ cells/mL: red). Different percentages of 0%, 10%, 30%, 50%, 70% and 100% conditioned media were applied before viral infection with FMDV O/SAU/18/2015 at a MOI of 0.01. Significance code: “****” p ≤ 0.0001, “***” p ≤ 0.001.

Figure 4

Increase or decrease of the cell density relative to the starting cell density. Increase or decrease in cell density after 20 h of viral infection at a cell density of (a) 1 × 10⁶ cells/mL or (b) 3 × 10⁶ cells/mL. Values were calculated from the values recorded in the experiment described above (Figure 3).

Figure 5

Cellular phenotype during batch cultivation. Cellular parameters such as (a) viable cell density (VCD) in cells/mL, (b) percent viability and (c) average diameter in µm were evaluated for BHK200 (orange), BHK300B (violet) and BHK300G (blue). Highlighted in white is the period of time in which viral infection and cellular virus production would take place at cell densities of 1 × 10⁶ cells/mL or 3 × 10⁶ cells/mL.

Figure 6

Calcium consumption of cells during batch analysis. Cells were grown in BHK200 (orange), BHK300B (violet) or BHK300G (blue) with either 100% medium supplementation or addition of 30% fresh medium.