2.1. Cell Lines and Culture Conditions
CHO K1 cells (Kao & Puck, 1968) (ATCC, Manassas, VA, USA) and CHO K1-derived cells adapted to serum-free suspension culture were used in this study. The CHO K1 cells were maintained in Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 (DMEM/F-12; Gibco, Paisley, UK) supplemented with 10% dialyzed fetal bovine serum (SAFC Biosciences, Lenexa, KS, USA). The CHO K1-derived, serum-free suspension-adapted cells were cultured in suspension using T-25 flasks (Greiner, Nürtingen, Germany) on a rotary shaker at 120 rpm (Taitec, Saitama, Japan). A total of 0.1×106/mL CHO cells were cultivated in 7 mL BalanCD CHO Growth A media (Irvine Scientific, Santa Ana, CA, USA). The suspension culture conditions were assessed using computational fluid and particle dynamics analyses with OpenFOAM version 2.3.0. (H. G. Weller, 1998). We modified the stirringInterPTFoam application to compute the culture conditions in box-shaped T-25 flasks on a rotary shaker (https://github.com/akionux/OpenFOAM-2.3.x/tree/master/applications/solvers/multiphase/stirringInterFoam/stirringInterPTFoam). All cells were cultivated at 37°C in a humidified atmosphere containing 5% CO2.