Immortalization of Human Adult Alveolar Epithelial Cells to Study Environmental Exposures of the Distal Lung

Alveolar epithelial cell type 1 (AT1) and type 2 (AT2) cells make up the saccular gas exchange units of the lung, called alveoli. Formation of alveoli during lung development accounts for the expansive surface area of the lung, allowing for proper respiration and delivery of oxygen to the body. Due to their delicate structure, alveoli are susceptible to injury caused by environmental exposures, such as inhaled cigarette smoke and heavy metals. Chronic exposure to these toxicants can exacerbate preexisting conditions such as asthma or contribute to the progression of lung diseases, such as chronic obstructive pulmonary disease (COPD), interstitial lung disease (ILD), and cancer. AT2 cells play a key role in injury repair and regeneration of the distal lung and there is widespread interest in their use as cellular models for lung disease in vitro. However, while immortalized cell lines derived from airway epithelial cells were successfully generated decades ago, human adult alveolar epithelial cell lines have proven to be more difficult to establish due to their limited proliferative capacity. Here we describe an extensive end-to-end method for deriving immortalized alveolar epithelial cells (AECs), termed "AEC-tLgT cells," from normal human lung tissue. We first outline a detailed procedure to isolate AT2 cells. We then outline our optimized method for immortalizing AT2 cells to generate polyclonal cell lines. We next describe a three-dimensional co-culture system to induce lung organoid formation from immortalized AT2 cell lines. Finally, we describe a procedure for studying cigarette smoke and nickel exposure using immortalized AT2 cell lines to investigate environmental toxicity. These protocols will enable users to generate AEC models from donors with defined genetic, demographic, and clinical backgrounds, facilitating the study of differential susceptibility to environmental exposures and risk for distal lung diseases. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation and purification of human AT2 cells Basic Protocol 2: Immortalization of AT2 cells and maintenance of resulting AEC lines Basic Protocol 3: Determination of organoid formation ability Basic Protocol 4: Treatment of immortalized AEC lines to study environmental exposures.