The physiological interactome of TCR-like antibody therapeutics in human tissues.

Selective binding of TCR-like antibodies that target a single tumour-specific peptide antigen presented by human leukocyte antigens (HLA) is the absolute prerequisite for their therapeutic suitability and patient safety. To date, selectivity assessment has been limited to peptide library screening and predictive modeling. We developed an experimental platform to de novo identify interactomes of TCR-like antibodies directly in human tissues using mass spectrometry. As proof of concept, we confirm the target epitope of a MAGE-A4-specific TCR-like antibody. We further determine cross-reactive peptide sequences for ESK1, a TCR-like antibody with known off-target activity, in human liver tissue. We confirm off-target-induced T cell activation and ESK1-mediated liver spheroid killing. Off-target sequences feature an amino acid motif that allows a structural groove-coordination mimicking that of the target peptide, therefore allowing the interaction with the engager molecule. We conclude that our strategy offers an accurate, scalable route for evaluating the non-clinical safety profile of TCR-like antibody therapeutics prior to first-in-human clinical application.

A multiplex inhalation platform to model in situ like aerosol delivery in a breathing lung-on-chip.

Prolonged exposure to environmental respirable toxicants can lead to the development and worsening of severe respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and fibrosis. The limited number of FDA-approved inhaled drugs for these serious lung conditions has led to a shift from in vivo towards the use of alternative in vitro human-relevant models to better predict the toxicity of inhaled particles in preclinical research. While there are several inhalation exposure models for the upper airways, the fragile and dynamic nature of the alveolar microenvironment has limited the development of reproducible exposure models for the distal lung. Here, we present a mechanistic approach using a new generation of exposure systems, the Cloud α AX12. This novel in vitro inhalation tool consists of a cloud-based exposure chamber (VITROCELL) that integrates the breathing AXLung-on-chip system (AlveoliX). The ultrathin and porous membrane of the AX12 plate was used to create a complex multicellular model that enables key physiological culture conditions: the air-liquid interface (ALI) and the three-dimensional cyclic stretch (CS). Human-relevant cellular models were established for a) the distal alveolar-capillary interface using primary cell-derived immortalized alveolar epithelial cells (AXiAECs), macrophages (THP-1) and endothelial (HLMVEC) cells, and b) the upper-airways using Calu3 cells. Primary human alveolar epithelial cells (AXhAEpCs) were used to validate the toxicity results obtained from the immortalized cell lines. To mimic in vivo relevant aerosol exposures with the Cloud α AX12, three different models were established using: a) titanium dioxide (TiO2) and zinc oxide nanoparticles b) polyhexamethylene guanidine a toxic chemical and c) an anti-inflammatory inhaled corticosteroid, fluticasone propionate (FL). Our results suggest an important synergistic effect on the air-blood barrier sensitivity, cytotoxicity and inflammation, when air-liquid interface and cyclic stretch culture conditions are combined. To the best of our knowledge, this is the first time that an in vitro inhalation exposure system for the distal lung has been described with a breathing lung-on-chip technology. The Cloud α AX12 model thus represents a state-of-the-art pre-clinical tool to study inhalation toxicity risks, drug safety and efficacy.

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 (AXiAEC) and organ-on-chip technology mimicking the lung alveolar biophysical environment (AXlung-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. AXiAECs 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, AXiAECs 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 ß1) 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 AXiAECs cultured on the AXlung-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 cm2) 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.

Second-generation lung-on-a-chip with an array of stretchable alveoli made with a biological membrane.

The air-blood barrier with its complex architecture and dynamic environment is difficult to mimic in vitro. Lung-on-a-chips enable mimicking the breathing movements using a thin, stretchable PDMS membrane. However, they fail to reproduce the characteristic alveoli network as well as the biochemical and physical properties of the alveolar basal membrane. Here, we present a lung-on-a-chip, based on a biological, stretchable and biodegradable membrane made of collagen and elastin, that emulates an array of tiny alveoli with in vivo-like dimensions. This membrane outperforms PDMS in many ways: it does not absorb rhodamine-B, is biodegradable, is created by a simple method, and can easily be tuned to modify its thickness, composition and stiffness. The air-blood barrier is reconstituted using primary lung alveolar epithelial cells from patients and primary lung endothelial cells. Typical alveolar epithelial cell markers are expressed, while the barrier properties are preserved for up to 3 weeks.

Impaired Wound Healing of Alveolar Lung Epithelial Cells in a Breathing Lung-On-A-Chip.

The lung alveolar region experiences remodeling during several acute and chronic lung diseases, as for instance idiopathic pulmonary fibrosis (IPF), a fatal disease, whose onset is correlated with repetitive microinjuries to the lung alveolar epithelium and abnormal alveolar wound repair. Although a high degree of mechanical stress (>20% linear strain) is thought to potentially induce IPF, the effect of lower, physiological levels of strain (5-12% linear strain) on IPF pathophysiology remains unknown. In this study, we examined the influence of mechanical strain on alveolar epithelial wound healing. For this purpose, we adopted the "organ-on-a-chip" approach, which provides the possibility of reproducing unique aspects of the in vivo cellular microenvironment, in particular its dynamic nature. Our results provide the first demonstration that a wound healing assay can be performed on a breathing lung-on-a-chip equipped with an ultra-thin elastic membrane. We cultured lung alveolar epithelial cells to confluence, the cells were starved for 24 h, and then wounded by scratching with a standard micropipette tip. Wound healing was assessed after 24 h under different concentrations of recombinant human hepatic growth factor (rhHGF) and the application of cyclic mechanical stretch. Physiological cyclic mechanical stretch (10% linear strain, 0.2 Hz) significantly impaired the alveolar epithelial wound healing process relative to culture in static conditions. This impairment could be partially ameliorated by administration of rhHGF. This proof-of-concept study provides a way to study of more complex interactions, such as a co-culture with fibroblasts, endothelial cells, or immune cells, as well as the study of wound healing at an air-liquid interface.

Medium throughput breathing human primary cell alveolus-on-chip model.

Organs-on-chips have the potential to improve drug development efficiency and decrease the need for animal testing. For the successful integration of these devices in research and industry, they must reproduce in vivo contexts as closely as possible and be easy to use. Here, we describe a 'breathing' lung-on-chip array equipped with a passive medium exchange mechanism that provide an in vivo-like environment to primary human lung alveolar cells (hAEpCs) and primary lung endothelial cells. This configuration allows the preservation of the phenotype and the function of hAEpCs for several days, the conservation of the epithelial barrier functionality, while enabling simple sampling of the supernatant from the basal chamber. In addition, the chip design increases experimental throughput and enables trans-epithelial electrical resistance measurements using standard equipment. Biological validation revealed that human primary alveolar type I (ATI) and type II-like (ATII) epithelial cells could be successfully cultured on the chip over multiple days. Moreover, the effect of the physiological cyclic strain showed that the epithelial barrier permeability was significantly affected. Long-term co-culture of primary human lung epithelial and endothelial cells demonstrated the potential of the lung-on-chip array for reproducible cell culture under physiological conditions. Thus, this breathing lung-on-chip array, in combination with patients' primary ATI, ATII, and lung endothelial cells, has the potential to become a valuable tool for lung research, drug discovery and precision medicine.

A microfluidic bubble trap and oscillator.

A new approach to trap air bubbles before they enter microfluidic systems is presented. The bubble trap is based on the combined interaction of surface tension and hydrodynamic forces. The design is simple, easy to fabricate and straightforward to use. The trap is made of tubes of different sizes and can easily be integrated into any microfluidic setup. We describe the general working principle and derive a simple theoretical model to explain the trapping. Furthermore, the natural oscillations of trapped air bubbles created in this system are explained and quantified in terms of bubble displacement over time and oscillation frequency. These oscillations may be exploited as a basis for fluidic oscillators in future microfluidic systems.

A lung-on-a-chip array with an integrated bio-inspired respiration mechanism.

We report a lung-on-a-chip array that mimics the pulmonary parenchymal environment, including the thin alveolar barrier and the three-dimensional cyclic strain induced by breathing movements. The micro-diaphragm used to stretch the alveolar barrier is inspired by the in vivo diaphragm, the main muscle responsible for inspiration. The design of this device aims not only at best reproducing the in vivo conditions found in the lung parenchyma but also at making the device robust and its handling easy. An innovative concept, based on the reversible bonding of the device, is presented that enables accurate control of the concentration of cells cultured on the membrane by easily accessing both sides of the membranes. The functionality of the alveolar barrier could be restored by co-culturing epithelial and endothelial cells that form tight monolayers on each side of a thin, porous and stretchable membrane. We showed that cyclic stretch significantly affects the permeability properties of epithelial cell layers. Furthermore, we also demonstrated that the strain influences the metabolic activity and the cytokine secretion of primary human pulmonary alveolar epithelial cells obtained from patients. These results demonstrate the potential of this device and confirm the importance of the mechanical strain induced by breathing in pulmonary research.

Electrically induced lipid migration in non-lamellar phase.

Inverted hexagonal blocks of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipid adsorbed on a polyethyleneimine (PEI)-coated surface in deionized water transformed its shape upon the application of an electric field, forming lipid objects in a variety of shapes (e.g. lines with a width of 10-50 µm). The phenomenon was driven by the electrophoresis, because the zwitterionic lipid, DOPE turned out to be highly negatively charged in deionized water. The interaction between DOPE and the PEI surface stabilized the system, assuring a lifetime over several weeks for the formed structures after the electric field was switched off. The free-drawing of microscopic objects (lines, crosses, and jelly fish) was also achieved by controlling the direction of the lipid movement with the field direction.