An improved 3D tetraculture system mimicking the cellular organisation at the alveolar barrier to study the potential toxic effects of particles on the lung

Abstract

BackgroundExposure to fine and ultra-fine ambient particles is still a problem of concern in many industrialised parts of the world and the intensified use of nanotechnology may further increase exposure to small particles. Complex in vitro coculture systems may be valuable tools to study particle-induced processes and to extrapolate effects of particles on the lung. A system consisting of four different human cell lines which mimics the cell response of the alveolar surface in vitro was developed to study native aerosol exposure (Vitrocell™ chamber). The system is composed of an alveolar type-II cell line (A549), differentiated macrophage-like cells (THP-1), mast cells (HMC-1) and endothelial cells (EA.hy 926), seeded in a 3D-orientation on a microporous membrane.ResultsThe spatial distribution of the cells in the tetraculture was analysed by confocal laser scanning microscopy (CLSM), showing a confluent layer of endothelial and epithelial cells on both sides of the transwell. Macrophage-like cells and mast cells can be found on top of the epithelial cells. The cells formed colonies under submerged conditions, which disappeared at the ALI. To evaluate the response to oxidative stress, the dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used together with 2,2’-azobis-2-methyl-propanimidamide-dihydrochloride (AAPH) as inducer of oxidative stress. The tetraculture showed less induction of reactive oxygen species (ROS) production after being treated with a positive control compared to the monocultures of EA.hy 926, THP-1 and HMC-1. Submerged cultures showed elevated ROS and IL-8 levels compared to ALI cultures. The Vitrocell™ aerosol exposure system was not significantly influencing the viability. Using this system, cells were exposed to an aerosol of 50 nm SiO2-Rhodamine NPs in PBS. The distribution of the NPs in the tetraculture after exposure was evaluated by CLSM. Fluorescence from internalized particles was detected in CD11b-positive THP-1 cells only.ConclusionThe system can be used in conjunction with a native aerosol exposure system and may finally lead to a more realistic judgement regarding the hazard of new compounds and/or new nano-scaled materials in the future. The results for the ROS production and IL-8 secretion suggest that submerged exposure may lead to an overestimation of observed effects.

The Fatty Acid Signaling Molecule cis -2-Decenoic Acid Increases Metabolic Activity and Reverts Persister Cells to an Antimicrobial-Susceptible State

“…In E. coli, exposure to cis-DA led to de novo production of proteins involved in transport (OppA), protein translation (TufA), metabolism (PoxB, FbaA, GadA, and SerC), and ATP synthesis and ATP hydrolysis (AtpA) (74)(75)(76)(77)(78)(79)(80). Proteins identified in E. coli under both cis-DA and saline conditions were involved in several functions, including metabolism (pyruvate dehydrogenase, AceF, and FrdA), ATP hydrolysis (polyphosphate kinase [PPK]), and protein repair (GroL) (81)(82)(83)(84)(85).…”

Preclinical safety and efficacy models for pulmonary drug delivery of antimicrobials with focus on in vitro models

“…These are microengineered biomimetic systems containing microfluidic channels lined by living human cells, which recreate specific components of lung physiology and pathology. The microfluid 3D lung-on-a-chip model showed to be more efficient to predict nanotoxicological effects, mainly due to the mechanical stress applied to the system [97]. Toxic and inflammatory responses as well as particle uptake were higher in this system compared to the conventional static monoculture.…”

“…Recently a series of 'organs-on-chips' have been produced specifically to meet the high-throughput needs for drugs and particle toxicity tests [97,98]. These are microengineered biomimetic systems containing microfluidic channels lined by living human cells, which recreate specific components of lung physiology and pathology.…”

Human lung epithelial cell cultures for analysis of inhaled toxicants: Lessons learned and future directions

“…Klein et al even extended this to a tetraculture model based on the Vitrocell aerosol exposure system using four different cell lines: AEC2 line (A549), macrophage-like cells (THP-1), a mast cell line (HMC-1) and an endothelial cell line (EA.hy 926) (Klein et al, 2013). Many studies on co-cultures focused on the use of continuous cell lines, because these can be more easily controlled and usually do not require cell-type specific media.…”