Figure 3.

Generation of primary human lung organoid-derived ALI cultures to study response to virus

(A) Schematic experimental design for primary human lung organoid-derived ALI culture generation: (1) cell expansion in submerged culture to obtain confluence at 100%; (2) initial differentiation in submerged cultures to foster tight junctions and barrier integrity, monitored by TEER values (>500 Ω cm²). (3) TEER goals are achieved, and cultures are transitioned to airlift by removal of apical media, which initiates final differentiation into pseudo-stratified epithelia. Cultures are monitored for a minimum of 4 weeks for the presence of beating cilia and mucus production.

(B) Representative photomicrographs of primary lung organoids-derived ALI cultures at Step 1 (3–4 days after seeding), Step 2 (at confluence approximately 12–14 days after seeding) and Step 3 (at 34 days post-airlift) captured using a bright-field microscope. Images were generated using ImageJ. Scale bars 500 μm, in black on the left corner.

(C) Measurement of trans-epithelial electrical resistance (TEER, Ω cm²) with error bars (mean ± SD), 3 measurements per time-point performed 3 times per week starting when the epithelium is confluent, one representative experiment per donor (four donors). Fully pseudo-stratified differentiated ALI cultures are obtained from primary lung organoid progenitors within 3–4 weeks (post-airlift). Note: Figure 3C reprinted with permission from Diana Cadena Castaneda et al. 2023¹ (Cell Press, Open Access).