# A human relevant in vitro alveolar epithelial barrier model to assess inhaled pollutant hazard

## TL;DR

This study creates a human-like lab model of lung alveolar cells to test the effects of inhaled pollutants, reducing the need for animal testing.

## Contribution

A triple cell co-culture model with human-relevant cell ratios and anatomical accuracy for in vitro toxicological testing of inhaled substances.

## Key findings

- The triple cell co-culture forms a tight alveolar barrier resembling human lung architecture.
- Macrophages in the model respond to inflammatory stimuli like lipopolysaccharide.
- The model can be used for high-throughput toxicological assessment of inhaled xenobiotics.

## Abstract

Relevant in vitro models could reduce the requirement for in vivo testing and allow higher throughput for imperative toxicological or pharmaceutical hazard testing. Models of the alveolar barrier are invaluable when assessing the toxicity of inhaled xenobiotics, though there is a requirement that these models are well-characterised and accurately resemble the relevant human cellular architecture. Here, a triple cell co-culture has been developed using hAELVi, NCI-H441 and differentiated THP-1 cells as models of type 1 and type 2 pneumocytes, and alveolar macrophages, respectively. Through pre-staining each cell type, confocal microscopy was first used to determine cell seeding ratios to hAELVi and NCI-H441 required to achieve a human-relevant 16.44:1 ± 3.29 ratio at the time of air-liquid interface exposure. CellTrackers were then used to ensure that the density of differentiated THP-1 cells was in line with previously published anatomical research at 1 cell/18 × 103 µm2. We were able to show that the triple culture forms a tight barrier and that the macrophages can respond to a (pro)-inflammatory stimulus (lipopolysaccharide). Given the anatomical relevancy and its ability to react to stimuli, this model may provide a useful platform to assess the toxicological hazard potential of a range of inhaled, respirable xenobiotics.

The online version contains supplementary material available at 10.1038/s41598-025-23768-4.