Development and Characterisation of an Immunocompetent Human In vitro Model of the Small Airways

Lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) increase the global health burden affecting hundreds of millions of people worldwide. The development of novel inhaled therapies is hindered by the lack of understanding of alveolar macrophages to inhaled particulate medicines. In animal models, there is increasing evidence that inhaled medicines are taken up by alveolar macrophages resident within the lung resulting in poorly understood perturbations. In vitro alveolar models are frequently employed to investigate the biological roles of alveolar macrophages in human health and disease and for inhaled drug discovery research. To generate translational data, it is crucial that such cell systems respond similarly to cells in situ. Cell culture lung models are available, but few are representative of alveolar drug delivery response, and, additionally, most exclude macrophages. In vitro cell culture models provide an ideal platform for assessing cell response to exposure of compounds in a high throughput manner and investigating the mechanisms involved that support undefined macrophage phenotype responses. To generate such platforms, it is first necessary to identify an appropriate, well-characterized cell line to develop a robust in vitro human model. To date, no human alveolar macrophage cell line is readily available. As such, current models rely on lung and blood-derived monocyte cell lines, which are differentiated using various stimuli to generate a mature monocyte/macrophage-like cell line that can be exposed to aerosols or particulate matter of interest. However, the methodologies available to differentiate and validate these cell lines are poorly defined, especially for U937 cells, whereby differentiation protocols vary significantly from the use of different stimuli, incubation and resting times. This research aimed to investigate if varying the differentiation protocols for U937 impacted the resulting cell characteristics and, with them, construct two co-culture models incorporating alveolar type I or type II epithelial cells to determine changes in alveolar like macrophage phenotypes in the presence of cationic amphiphilic drug amiodarone commonly used to generate a 'foamy' macrophage phenotype. Our results suggested that the concentration of PMA U937 cells are exposed to has a more significant impact on cell characteristics (differentiation markers and functionality) than the length of incubation. A differentiation protocol was adopted, and co-culture models were characterized regarding their morphology and barrier properties, with permeability assays and cytokine interactions. High content image analysis assessed macrophage responses when exposed to drug concentrations of amiodarone. Morphological changes induced by amiodarone could be compared rather than determined cell death for vacuolated "foamy" macrophage assessments. In summary, these models were characterised regarding their morphology and viability properties, with functional assays showing applicability for evaluating macrophage cell response and cell-cell communication. Cocultivation allows a more physiologically relevant representation of the lower airway, and immunocompetent in vitro models can be applied as a valuable tool to evaluate new formulations intended for pulmonary delivery