Tonsil Organoid Culture and Treatment

Tonsil organoids were established as previously described (68 (link), 69 (link)). Briefly, whole tonsils (overall healthy, without obvious signs of inflammation) were collected in saline after surgery and then immersed in an antimicrobial bath of Ham’s F12 medium (Gibco) containing Normocin (InvivoGen), penicillin, and streptomycin for 1 h at 4 °C for decontamination. Tonsils were then briefly rinsed with PBS and manually disrupted into a suspension by processing through a 100 μm strainer with a syringe plunger and cryopreserved. Frozen cells were thawed, washed, enumerated, and then plated (6 × 10⁶ cells in 100 μL per well) into permeable (0.4 μm pore size) membranes placed in standard 12-well tissue-culture plates. Organoids were cultured for 7 d at 37 °C, 5% CO₂ with or without Mtb-lysate (10 μg/mL, BEI Resources) or LAIV (1 μL per well, an equivalent of 1.6 × 10⁴ to 1.6 × 10⁵ fluorescent focus units per strain; FluMist Quadrivalent, Medimmune). Harvested cells were washed followed by flow cytometry analysis.