Air-Liquid Interface Culture Model to Study Lung Cancer-Associated Cellular and Molecular Changes

Airway epithelial cells arrayed in the inner lining of the airways of the lung are believed to be the major source for the development of malignancy of the lung. The advent of in vitro cell culture model made it easy to understand the molecular mechanism of carcinogenesis at a cellular level, where the airway epithelial cells are cultured on a 2D surface submerged in the culture media. However, this method of culturing airway epithelial cells does not reflect their true nature, and thus results obtained have their limitations. Further, they exhibit dissimilar morphology, transcriptome, and secretome when compared to the cells in vivo. Thus, the experimental data obtained from 2D culture models are inconclusive and, in most cases, could not be validated further in in vivo settings. These limitations can be addressed by culturing the airway epithelial cells on air-liquid interface (ALI), where they develop ciliated morphology similar to that of the lung. Experiments performed with this 3D model provide reliable data that are more realistic, and, in many cases, could replace the requirement of further in vivo validation. Here, we provide the detailed protocol of a 3D culture system called ALI culture for growing human-derived primary small airway epithelial cells to study the cellular and molecular changes associated with lung cancer.