Generating Tumor Organoid Cultures from the Lung Adenocarcinoma Cell Line

Here, we describe a protocol for generating Cell Line-Derived Tumor Organoids (CDTOs) from the A549 human lung adenocarcinoma cell line. The protocol involves embedding 2D-expanded A549 cells in Matrigel and maintaining them in 3D culture medium for long-term culture. Recommended seeding density of 500 cells/µL was determined to support consistent organoid formation. The resulting CDTOs were characterized by hematoxylin and eosin (H&E) staining and immunofluorescence (IF). The organoids maintained high expression of the lung adenocarcinoma markers Thyroid Transcription Factor 1 (TTF-1), adhesion protein E-Cadherin (ECAD), and cytoskeleton protein Keratin 7 (KRT7). Furthermore, tight junction protein Zona Occludens 1 (ZO-1) expression showed dysregulated polarity of tumor organoids. This protocol offers a technically straightforward, cost-effective, and purely tumorous organoid platform for lung adenocarcinoma research. Its simplicity and reproducibility also make it suitable for undergraduate laboratory teaching, where it can help students acquire fundamental 3D tumor organoid culture techniques within a limited lab schedule.