Cell culture
Proprietary suspension adapted Human Embryonic Kidney 293 (HEK293) cells were provided by REGENXBIO (Rockville, MD). Cells were cultured in a serum free medium supplemented with L-glutamine (Thermo Scientific, Waltham, MA). Cells were maintained in an orbital shaking incubator (Infors, Bottmingen, Switzerland) at 30 mL culture volumes in 125 mL Erlenmeyer flasks (Corning) at 37°C, 5% CO2 and 85% humidity, with agitation at 140 rpm. Smaller scale cultures were grown in shallow-well 24-well plates (0.7 mL culture volume/well, 240 rpm shaking) (Corning, Acton, MA) using the Deutz system[14]. Routine cell density and cell viability measurements were performed on a ViCell automated cell counter (Beckman-Coulter, Brea, CA). Measurements of density and viability and determination of mean cell volume during rAAV production runs were performed using the Norma HT system (Iprasense) utilising 3 µl total cell culture and 20 µm slide chambers. Mean cell volume (V ) was calculated as \(V=\frac{4}{3}\pi r^{3}\) where r = measured cell diameter/2.