High-Titer Hepatitis C Virus Production in a Scalable Single-Use High Cell Density Bioreactor

2. Materials and Methods

2.1. Maintenance of Huh7.5 Cells

Huh7.5 cell cultures were maintained in cell culture T flasks in serum-containing medium (SCM): Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Paisley, UK) containing 4 mM GlutaMAX and supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Sigma). In T175 flasks (Nunc, Roskilde, Denmark) and triple layer culture flasks (Nunc), Huh7.5 cells were passaged every 2–3 days. All cell cultures were maintained at 37 °C and 5% CO2.

2.2. HCV Virus Stocks

Two sequence-confirmed stocks of the genotype 5a recombinant SA13/JFH1core-NS5B [28], grown in monolayer Huh7.5 cell cultures in T flasks, were used in this study. A third passage virus stock with an infectivity titer of 5.8 log10 FFU/mL, grown under serum-containing (SC) conditions [39], was used to inoculate CelCradle™ cultures, shake flask cultures, and for virus characterization experiments. A fourth passage virus stock with an infectivity titer of 5.1 log10 FFU/mL [32], grown under SF conditions [39], was used for virus characterization experiments.

Source from: https://www.mdpi.com/2076-393X/10/2/249/htm