Air-liquid interface (ALI) epithelial cell (EC) cultures are useful for studying the effect of exposure to airborne substances, but require substantial cell numbers that cannot always obtained from patients. Recent studies show that 3D organoid cultures can be initiated from samples with limited EC numbers, such as BAL (Sachs et al, 2019). Aim of the present study was to compare expansion of EC using 3D organoid culture to conventional 2D plastic-basic expansion for establishment of ALI cultures, and whether such cultures can be used for exposure to cigarette smoke (CS). Airway EC were obtained from tracheal aspirates (TA) from preterm newborns and bronchoalveolar lavage (BAL), or from bronchial tissue (BT) from adults. TA and BAL cells were 3D-expanded, whereas cells from BT were expanded in 3D and 2D. Following expansion, EC were cultured at ALI to induce differentiation. The impact of cell origin and 2D or 3D expansion was assessed with respect to (i) cellular composition; (ii) response to CS exposure; (iii) effect of Notch inhibition or IL-13 stimulation on cellular differentiation. Well-differentiated ALI cultures were established from all samples and cellular compositions were comparable. All 3D-expanded cultures showed a similar oxidative stress and unfolded protein response following CS exposure, but different from the 2D-expanded cultures. Additionally, TA- and BAL-derived cultures were less sensitive to modulation by DAPT or IL-13 than BT-derived cultures. We conclude that organoid-based expansion of clinical samples with low epithelial cell numbers, such as tracheal aspirates from preterm newborns, is a valid method and tool to establish ALI cultures.