26 An Advanced Triple Cell co-Culture of the Alveolar Epithelial Barrier for Inhalation Hazard Assessment in Vitro

Abstract

In vitro lung models are a valuable tool to assess the inhalation hazard of air pollutants, bioaerosols, chemicals and nanomaterials. Though it is essential that these models are characterised and anatomically relevant to the area of the human lung they seek to represent. Previously within inhalation toxicology, focus has been placed on the use of monocultures of alveolar epithelial cells, however use of alveolar co-culture models have been presented as more realistic and sensitive models. Here, we present the development of a novel triple cell co-culture system, cultured at the air-liquid interface that is sensitive to LPS and particulate matter. This co-culture contains alveolar type 1 cells (hAELVi), alveolar type 2 cells (NCI-H441) and alveolar macrophages (differentiated THP-1 (dTHP-1) using PMA (20 nM)). Using CellTrackers™ with confocal microscopy, the ratio of hAELVi cells to NCI-H441 cells was confirmed to be a histologically relevant 16:1 ratio forming a tight monolayer shown through blue dextran membrane permeability assay. Macrophages were seeded on top of the epithelial co-culture at an anatomically relevant density of 1 macrophage per 18x103 μm2 (confirmed via confocal microscopy) to form the triple cell co-culture. The model, when challenged, released more interleukin-8 and TNF-α than the epithelial mono- and bi-culture systems when exposed to LPS (1 μg/mL for 24 hrs) (p<0.05), suggesting that the macrophages do mount the theoretical (pro-)inflammatory response. This anatomically relevant in vitro model forms the basis to investigate inhaled exogenous substances, such as the differential toxicology impacts of particulate matter and nitrogen dioxide.