Robust, physiologically relevant in vitro lung model systems are required to properly investigate respiratory physiology and pathology. However, obtaining truly differentiated air-liquid-interface (ALI) cultures with airway epithelial cells can be challenging, due to the limited expansion and differentiation potential associated with primary cells. In this virtual paper presentation, Dr. Rachael Rayner evaluates the effects of different expansion media on the ability to expand primary normal human bronchial epithelial cells while maintaining their 3D culture characteristics. Her research highlights the need for more critical assessments of expansion and differentiation culture conditions to obtain optimal morphology and functional readouts.