Impact of Influenza A Virus Infection on Growth and Metabolism of Suspension MDCK Cells Using a Dynamic Model

 Materials and Methods

3.1. Shake Flask Cultivations

Pre-cultures of MDCK.SUS2 suspension cells were grown in shaker flasks (125 mL polycarbonate Erlenmeyer flasks, #431143, Corning®, New York city, NY, USA) with 50 mL working volume (wv), in a Multitron Pro incubator (Infors HT, Bottmingen, Switzerland) at 37 °C and 5% CO2 atmosphere with a shaking frequency of 180 rpm. Cells were passaged every 3–4 days with a seeding density of 0.5–0.8 × 106 cells/mL.

IAV A/Puerto Rico/8/34 (H1N1) seed virus was used for infection, generated in adherent MDCK cells (ECACC # 84121903). The seed virus had a titer of 1.1 × 109 TCID50/mL.

Cells were cultivated in a chemically defined, protein-free and animal component-free medium, Smif8 (Smif8 PGD 2×, supplemented with 5 mM glutamine, and 8 mM pyruvate), specifically developed for the cultivation of suspension MDCK cells [95].

Growth and infection experiments for model validation were performed using 500 mL shaking flasks (#4113-0500, Nalgene™, Thermo Scientific, Waltham, MA, USA) with an initial cultivation volume of 200 mL at 150 rpm. Two cultivations were performed, one mock-infection (Cultivation 1), where cells grew for about 200 h, and one infection (Cultivation 2), where cells were infected with IAV at about 49 h post inoculation. To achieve a synchronous infection of the cell population, a moi = 10 was used. Due to the relatively low cell concentration at time of infection (2.1 × 106 cells/mL) and the high moi used, neither medium replacement nor trypsin addition (for virus activation) was necessary. In both cultivations, sterile Milli-Q water was added before sampling to compensate for water evaporation (1–2 mL/day) since the experiment was performed in a non-hydrated incubator.

Source from: https://www.mdpi.com/2218-1989/12/3/239/htm