Advancing alveolar in-vitro research with cell-line based air-liquid interface cultures

Sammanfattning

Alveoli are sac-like structures in the distal lung specialised for gaseous exchange. Alveolar epithelium facilitates efficient respiration through a thin and robust arrangement of type-I (AT1) and type-II (AT2) pneumocytes, supported by tight junctions and surfactant-rich alveolar lining fluid. Chronic diseases like pulmonary fibrosis, asthma, and sarcoidosis compromise alveolar integrity, resulting in dysfunctions such as inflammation, edema, emphysema, or airway remodelling. In-vitro models are essential to understanding these molecular mechanisms in health and disease. This study investigated the feasibility of four commonly used alveolar epithelial cell models in air-liquid interface (ALI) cultures. These included adenocarcinoma-derived A549 and H441 cells, and immortalised primary cell-lines Aelvi and Arlo. The models were evaluated for barrier formation, alongside phenotypic characterisation, and functional assessments. H441, Aelvi, and Arlo cells maintained a functional barrier for up to three weeks and expressed alveolar epithelium associated markers under ALI conditions. Despite transformation to serum-free media, A549 cells failed to develop a barrier and were not characterised further in this study. Treatment with inflammatory and fibrotic stimuli reduced the transepithelial electrical resistance (TEER) and triggered release of interleukin-6 (IL6), Granulocyte-macrophage-colony stimulating factor (GM-CSF), interleukin-8 (IL8), and Chemokine (C-C motif) ligands - CCL5, CCL17, and CCL22, indicating onset of inflammatory response. Additionally, fibrotic treatment induced gene markers of aberrant basaloid differentiation as well as epithelial-mesenchymal transition, indicating potential to mimic fibrosis-driven tissue remodelling. The H441 model demonstrated AT2-like characteristics with surfactant expression and inflammatory responsiveness; Aelvi showed both AT1 and AT2 markers with pronounced fibrotic response; and Arlo cells developed a reactive TEER relevant for studying barrier function. These findings demonstrate the strengths and limitations of different cell-line–based ALI models in studying alveolar biology. Further studies can explore their long-term stability and translational application. This would enable model matching to specific research needs, thus improving the relevance and impact of in-vitro ALI models in alveolar research.