Abstract
A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor α) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.
Highlights
In vitro lung cell cultures offer cost-effective, robust, and well-controlled platforms to assess the hazards of aerosols[1]
It has been shown that when the cells are cultured at an air-liquid interface (ALI), surfactant is released on the apical side of air-exposed epithelial cells, reducing surface tension[11,12,13]
The aim of this study is to demonstrate the 3D human alveolar epithelial tissue model assembly, including the isolation of peripheral blood monocytes (PBMs) from human buffy coats
Summary
In vitro lung cell cultures offer cost-effective, robust, and well-controlled platforms to assess the hazards of aerosols[1]. As a model cell system for human alveolar pneumocytes, the epithelial A549 cell line isolated from a pulmonary adenocarcinoma is often used[2] These cells represent squamous type II epithelial cells from the alveolar region[3] and are a widely used lung cell line for hazard and toxicity assessment[1,4,5,6,7,8,9,10]. It has been shown that when the cells are cultured at an air-liquid interface (ALI), surfactant is released on the apical side of air-exposed epithelial cells, reducing surface tension[11,12,13] This feature is important in nanomaterial respiratory hazard and toxicity investigations. Even though A549 cells form a monolayer (which can overgrow into multilayers at later timepoints when cultivated at ALI) and produce surfactant, a drawback is their insufficient tight junction formation, resulting in low transepithelial electrical resistance values, but still presenting a functional barrier against intercellular (nano)particles translocation[16,17,18]
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