2.5 Chinese Hamster Ovary (CHO) Cell Expansion
CHO-S cells (Gibco R80007) were cultured in Freestyle CHO expression medium (Gibco 12651014) supplemented with 8 mM L-glutamine (Gibco 25030081), 0.1% Kolliphor P188 (Sigma 15759), and 0.5X Pen/Strep (Gibco 15140122). Cells (passage 3-7) were transferred from active culture in 125-mL shake flask (DWK Life Sciences WPFPC0125S) and seeded in RBL bioreactor 96-well microplate at a concentration of 150,000 cells/mL per well with a volume of 300 µL. The plates were then sealed with a breathable rayon-acrylate film (Thermo Scientific™ 241205) to prevent evaporative loss during culture. Drive system was set to duty-cycle (10 min. ON, 50 min. OFF) at a selected rate (6000 RPM) and placed inside a cell culture incubator at 37oC with a humidified atmosphere at 8% CO2.  Total protein values for each culture condition at the end of a 72-h culture period were determined by Bradford protein assay kit (Thermo Scientific™ 23246) following complete cell lysis. The resulting protein values were reported as cell concentration (cells/mL) after comparison to standard protein curve obtained from known CHO-S cell counts.