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

Abstract

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.

Popular Abstract

Cell based models that mimic the alveoli for studying inflammation and fibrosis.

Alveoli are the primary site for gaseous exchange. Embedded deep within the lungs, they assume a thin yet robust architecture. Together with the surrounding cells from blood capillaries, alveolar cells form the air-blood barrier. This interface allows respiratory gases to travel between the air-space in lungs and blood in the capillaries.

The constant process of gaseous exchange is made possible by a combined function of many physical and structural components in the alveolar environment. The extracellular matrix and fibroblasts from the alveolar interstitial space, as well as the lining fluid inside the alveoli ensure their stability. Any disruptions to this complex organisation can result in serious dysfunctions as seen in many lung diseases like chronic obstructive pulmonary disorder, idiopathic pulmonary fibrosis and acute respiratory distress syndrome. This necessitates the study of alveoli in health and disease, probing a great need for reliable tools to mimic the alveolar structure and functions outside the human body.

Although the complexity of alveolar architecture is hard to replicate in the lab, this study shows that different cell-lines can demonstrate specific alveolar functions. We grew the cells on top of a permeable membrane and provided media from below the cell layer. The upper portion remained exposed to air, forming an air-liquid interface. This replicated the natural environment to facilitate growth of compact cell layers as seen in the alveoli. Once fully grown, the cell layers formed a strong barrier like the alveoli whose strength could be monitored through measuring the transepithelial electrical resistance.

After comparative evaluation of four cell lines in air-liquid interface, this study characterised three functional systems with different strengths and disease modelling capacity. The cancerous H441 cells formed thick and compact layers. This model showed strong presence of surfactant producing cells in the alveoli and released cytokines as seen in acute phase inflammation related to COPD and Asthma. Immortalised Arlo cells developed a highly responsive barrier suitable for studying structural disruptions in conditions like emphysema where the walls between the alveoli are disrupted. Finally, Aelvi cells, which are the mother cell line of Arlo, formed the strongest barrier and showed strong potential as a tool to study fibrosis.

This study emphasizes the importance of culturing technique in successfully mimicking the alveolar characters. A major strength of this work lies in findings that clarify the suitability of different cell systems in representing different disease conditions. With more characterisations of their genome and proteome, their comparative strengths can be further established. Nevertheless, this work forms the basis for creating flexible models that can be adapted to study different diseases and test therapeutic compounds.

Master’s Degree Project in Molecular Biology 60 credits 2025
Department of Biology, Lund University
Advisor: Jenny Horndahl, Linda Yrlid
Respiratory and Immunology - COPD-IPF, AstraZeneca R&D

• author: Dambhare, Setu
• supervisor: Jenny Horndahl; Lind Yrlid
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20242
• year: 2025
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9218299
• date added to LUP: 2026-01-12 10:20:42
• date last changed: 2026-01-12 10:20:42