Tonsillar Mononuclear Cell Organoid Assay

Excised tonsils from three de-identified immunocompetent patients were diced and strained through a 100-μm filter. A single cell suspension of tonsillar mononuclear cells (MNCs) was created with Ficoll density gradient separation. Once isolated, MNC were counted and resuspended in organoid media (RPMI with L-glutamine, 10% FBS, 2 mM glutamine, 1X penicillin-streptomycin, 1 mM sodium pyruvate, 1X MEM non-essential amino acids, 10 mM HEPES buffer and 1 μg/ml of recombinant human B cell activating factor [BioLegend]) at a concentration of 6 × 10⁷ cells per ml. As previously described by Wagar et al. [32 (link)], MNC’s were transferred to permeable transwells (0.4-μm pore, 12-mm diameter; Millipore). Transwells were inserted into standard 12-well polystyrene plates containing 1 ml of additional organoid media and placed in an incubator at 37 °C and 5% CO2. Lometrexol in phosphate-buffered saline was added, or not, to organoids after 48 h in culture. Organoid media with or without lometrexol was replaced every 3 days. On culture day 7, organoid MNCs were resuspended and stained at 4 °C with the anti-human CD38 (HIT2; BioLegend), CD27 (O323; BioLegend), CD19 (HIB19; BioLegend), and L/D aqua (Invitrogen). Cells were analyzed with LSRFortessa (BD Bioscience) and visualized with FlowJo software (TreeStar).