Cell Density Plays a Critical Role in Ex Vivo Expansion of T Cells for Adoptive Immunotherapy

Figure 4

Reactive oxygen species (ROS) are the mediators of ATC apoptosis at low cell density. (a) Intracellular ROS levels in ATCs cultured at different cell densities. ATCs were cultured at high cell density (1×10⁵/mL) or low cell density (1×10⁴/mL) in 75 cm² flasks for 24 hours. After staining with DHR for 30 minutes, intracellular ROS levels of ATCs were analyzed by flow cytometry. Nonstaining ATCs were used as negative control. (b) Antioxidants protect activated T cells from apoptosis at low cell density. ATCs were cultured at 1×10⁴/mL in 75 cm² flasks in the presence of catalase, NAC, and human serum albumin (HSA) in different concentrations. HSA in unsupplemented serum-free medium was measured at 3 mg/mL by Bradford assay. Both cell viability and intracellular ROS levels were analyzed by flow cytometry as in (a). ROS levels were indicated as mean fluorescence intensity (MFI) of DHR-stained cells. Similar results were obtained in 2 independent experiments. (c) Elevated intracellular albumin in ATCs cultured at low cell density. ATCs were cultured at high cell density (1×10⁵/mL) or low cell density (1×10⁴/mL) in 75 cm² flasks for 24 hours. Cells were fixed and permeabilized and then stained with mouse antihuman albumin antibody, followed by staining with a secondary goat antimouse FITC-conjugated antibody. Cells stained with only secondary antibodies were used as negative controls.