Microfluidic acini-on-chip platforms as a tool to study bacterial lung exposure

Bacterial invasion of the respiratory system leads to complex immune responses involving many cell types. In the alveolar regions, the first line of defense includes the alveolar epithelium, secreted surfactant, alveolar lining fluid and alveolar macrophages. The epithelium consists of alveolar type I and type II cells. Both cell types are known to have immuno-modulatory functions characterized by the secretion of pro-inflammatory cytokines. Epithelial in vitro models offer attractive platforms to investigate biological functionality, but have typically relied on traditional well plate assays that come short of mimicking the complexity of the airway environment and do not capture physiological flows or relevant anatomical features. In the last decade, microfluidics have gained significant momentum in laying the foundations for constructing in vitro models that mimic physiologically-relevant organ functions. Here we propose to use acinus-on-chip platforms that mimic more closely native acinar microflows at true scale in a multi-generation alveolated tree. Acinar chips are cultured with human Alveolar Epithelial Lentivirus immortalized (hAELVi) cells at an air-liquid interface (ALI); such cells show alveolar type I like characteristics and maintained barrier function, leading to high trans-epithelial electrical resistance (TEER) in analogy to primary cells harvested from human tissue. To model bacterial infection, i.e. a strong stimulator of the innate arm of the immune system, lipopolysaccharides (LPS) will be used. LPS is a major outer surface membrane protein expressed on Gram-negative bacteria. The alveolar epithelium is exposed to LPS-laden aerosols and cell response is monitored mainly by secretion of pro-inflammatory cytokines. Our acinus-on-chip allows quantitative on-line measurements of alveolar barrier function, absorption kinetics and immunologically relevant responses, giving further insight to the role played by type I alveolar cells in lung immunity. Please click Additional Files below to see the full abstract

Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells.

Vukosavljevic B, Hittinger M, Hachmeister H, et al. Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells. Journal of Biophotonics. 2019;12(6): e201800052

An Outer Membrane Vesicle-Based Permeation Assay (OMPA) for Assessing Bacterial Bioavailability

When searching for new antibiotics against Gram-negative bacterial infections, a better understanding of the permeability across the cell envelope and tools to discriminate high from low bacterial bioavailability compounds are urgently needed. Inspired by the phospholipid vesicle-based permeation assay (PVPA), which is designed to predict non-facilitated permeation across phospholipid membranes, outer membrane vesicles (OMVs) of Escherichia coli either enriched or deficient of porins are employed to coat filter supports for predicting drug uptake across the complex cell envelope. OMVs and the obtained in vitro model are structurally and functionally characterized using cryo-TEM, SEM, CLSM, SAXS, and light scattering techniques. In vitro permeability, obtained from the membrane model for a set of nine antibiotics, correlates with reported in bacterio accumulation data and allows to discriminate high from low accumulating antibiotics. In contrast, the correlation of the same data set generated by liposome-based comparator membranes is poor. This better correlation of the OMV-derived membranes points to the importance of hydrophilic membrane components, such as lipopolysaccharides and porins, since those features are lacking in liposomal comparator membranes. This approach can offer in the future a high throughput screening tool with high predictive capacity or can help to identify compound- and bacteria-specific passive uptake pathways

A New Immortalized Human Alveolar Epithelial Cell Model to Study Lung Injury and Toxicity on a Breathing Lung-On-Chip System

The evaluation of inhalation toxicity, drug safety and efficacy assessment, as well as the investigation of complex disease pathomechanisms, are increasingly relying on in vitro lung models. This is due to the progressive shift towards human-based systems for more predictive and translational research. While several cellular models are currently available for the upper airways, modelling the distal alveolar region poses several constraints that make the standardization of reliable alveolar in vitro models relatively difficult. In this work, we present a new and reproducible alveolar in vitro model, that combines a human derived immortalized alveolar epithelial cell line ((AX)iAEC) and organ-on-chip technology mimicking the lung alveolar biophysical environment ((AX)lung-on-chip). The latter mimics key features of the in vivo alveolar milieu: breathing-like 3D cyclic stretch (10% linear strain, 0.2 Hz frequency) and an ultrathin, porous and elastic membrane. (AX)iAECs cultured on-chip were characterized for their alveolar epithelial cell markers by gene and protein expression. Cell barrier properties were examined by TER (Transbarrier Electrical Resistance) measurement and tight junction formation. To establish a physiological model for the distal lung, (AX)iAECs were cultured for long-term at air-liquid interface (ALI) on-chip. To this end, different stages of alveolar damage including inflammation (via exposure to bacterial lipopolysaccharide) and the response to a profibrotic mediator (via exposure to Transforming growth factor β1) were analyzed. In addition, the expression of relevant host cell factors involved in SARS-CoV-2 infection was investigated to evaluate its potential application for COVID-19 studies. This study shows that (AX)iAECs cultured on the (AX)lung-on-chip exhibit an enhanced in vivo-like alveolar character which is reflected into: 1) Alveolar type 1 (AT1) and 2 (AT2) cell specific phenotypes, 2) tight barrier formation (with TER above 1,000 Ω cm(2)) and 3) reproducible long-term preservation of alveolar characteristics in nearly physiological conditions (co-culture, breathing, ALI). To the best of our knowledge, this is the first time that a primary derived alveolar epithelial cell line on-chip representing both AT1 and AT2 characteristics is reported. This distal lung model thereby represents a valuable in vitro tool to study inhalation toxicity, test safety and efficacy of drug compounds and characterization of xenobiotics

Calcifediol-loaded liposomes for local treatment of pulmonary bacterial infections.

The influence of vitamin D3 and its metabolites calcifediol (25(OH)D) and calcitriol on immune regulation and inflammation is well described, and raises the question of potential benefit against bacterial infections. In the current study, 25(OH)D was encapsulated in liposomes to enable aerosolisation, and tested for the ability to prevent pulmonary infection by Pseudomonas aeruginosa. Prepared 25(OH)D-loaded liposomes were nanosized and monodisperse, with a negative surface charge and a 25(OH)D entrapment efficiency of approximately 23%. Jet nebulisation of liposomes was seen to yield an aerosol suitable for tracheo-bronchial deposition. Interestingly, 25(OH)D in either liposomes or ethanolic solution had no effect on the release of the proinflammatory cytokine KC from Pseudomonas-infected murine epithelial cells (LA-4); treatment of infected, human bronchial 16-HBE cells with 25(OH)D liposomes however resulted in a significant reduction in bacterial survival. Together with the importance of selecting an application-appropriate in vitro model, the current study illustrates the feasibility and practicality of employing liposomes as a means to achieve 25(OH)D lung deposition. 25(OH)D-loaded liposomes further demonstrated promising effects regarding prevention of Pseudomonas infection in human bronchial epithelial cells

Synthesis and characterization of human transferrin-stabilized gold nanoclusters.

Human transferrin has been biolabelled with gold nanoclusters (Au NCs) using a simple, fast and non-toxic method. These nanocrystals (<2 nm) are stabilized in the protein via sulfur groups and have a high fluorescence emission in the near infrared region (QY=4.3%; λem=695 nm). Structural investigation and photophysical measurements show a high population of clusters formed of 22-33 gold atoms covalently bound to the transferrin. In solutions with pH ranging from 5 to 10 and in buffer solutions (PBS, HEPES), those biolabelled proteins exhibit a good stability. No significant quenching effect of the fluorescent transferrin has been detected after iron loading of iron-free transferrin (apoTf) and in the presence of a specific polyclonal antibody. Additionally, antibody-induced agglomeration demonstrates no alteration in the protein activity and the receptor target ability. MTT and Vialight® Plus tests show no cytotoxicity of these labelled proteins in cells (1 µg ml(-1)-1 mg ml(-1)). Cell line experiments (A549) indicate also an uptake of the iron loaded fluorescent proteins inside cells. These remarkable data highlight the potential of a new type of non-toxic fluorescent transferrin for imaging and targeting

Macrophage uptake of cylindrical microparticles investigated with correlative microscopy.

Cylindrical particles offer the opportunity to develop controlled and sustained release systems for the respiratory tract. One reason is that macrophages can phagocyte such particles only from either of the two ends. We investigated the uptake behaviour of murine alveolar macrophages incubated with elongated submicron-structured particles. For that purpose, fluorescent model silica nanoparticles were interconnected with the biocompatible polysaccharide agarose, building up cylindrical particles within the pores of track-etched membranes. In contrast to common approaches we determined the uptake at different time points with scanning electron microscopy, fluorescence microscopy, and the combination of both techniques - correlative microscopy (CLEM). As a consequence, we could securely identify uptake events and observe in detail the engulfment of particles and confirm, that phagocytosis could only be observed from the tips of the cylinders. CLEM allowed a comparison of the uptake measured with different techniques at identical macrophages. Qualitative and quantitative evaluation of this cylindrical particle uptake showed substantial differences between fluorescence microscopy, electron microscopy and the combination of both (CLEM) within 24h

Editorial EJPB - Biobarriers 2018.

[No abstract available

Barriers and motivations for non-invasive drug delivery.

Editorial anlässlich der Special Issue zu unserer BioBarriers Konferenz von 2016