Development of a dynamic in vitro stretch model of the alveolar interface with aerosol delivery.

We describe the engineering design, computational modeling, and empirical performance of a moving air-liquid interface (MALI) bioreactor for the study of aerosol deposition on cells cultured on an elastic, porous membrane which mimics both air-liquid interface exposure conditions and mechanoelastic motion of lung tissue during breathing. The device consists of two chambers separated by a cell layer cultured on a porous, flexible membrane. The lower (basolateral) chamber is perfused with cell culture medium simulating blood circulation. The upper (apical) chamber representing the air compartment of the lung is interfaced to an aerosol generator and a pressure actuation system. By cycling the pressure in the apical chamber between 0 and 7 kPa, the membrane can mimic the periodic mechanical strain of the alveolar wall. Focusing on the engineering aspects of the system, we show that membrane strain can be monitored by measuring changes in pressure resulting from the movement of media in the basolateral chamber. Moreover, liquid aerosol deposition at a high dose delivery rate (>1 µl cm-2 min-1 ) is highly efficient (ca. 51.5%) and can be accurately modeled using finite element methods. Finally, we show that lung epithelial cells can be mechanically stimulated under air-liquid interface and stretch-conditions without loss of viability. The MALI bioreactor could be used to study the effects of aerosol on alveolar cells cultured at the air-liquid interface in a biodynamic environment or for toxicological or therapeutic applications.

Biomimetic In Vitro Lung Models: Current Challenges and Future Perspective.

As post-COVID complications, chronic respiratory diseases are one of the foremost causes of mortality. The quest for a cure for this recent global challenge underlines that the lack of predictive in vitro lung models is one of the main bottlenecks in pulmonary preclinical drug development. Despite rigorous efforts to develop biomimetic in vitro lung models, the current cutting-edge models represent a compromise in numerous technological and biological aspects. Most advanced in vitro models are still in the "proof-of-concept" phase with a low clinical translation of the findings. On the other hand, advances in cellular and molecular studies are mainly based on relatively simple and unrealistic in vitro models. Herein, the current challenges and potential strategies toward not only bioinspired but truly biomimetic lung models are discussed.

Bioactive Cell-Derived ECM Scaffold Forms a Unique Cellular Microenvironment for Lung Tissue Engineering.

Chronic lung diseases are one of the leading causes of death worldwide. Lung transplantation is currently the only causal therapeutic for lung diseases, which is restricted to end-stage disease and limited by low access to donor lungs. Lung tissue engineering (LTE) is a promising approach to regenerating a replacement for at least a part of the damaged lung tissue. Currently, lung regeneration is limited to a simplified local level (e.g., alveolar−capillary barrier) due to the sophisticated and complex structure and physiology of the lung. Here, we introduce an extracellular matrix (ECM)-integrated scaffold using a cellularization−decellularization−recellularization technique. This ECM-integrated scaffold was developed on our artificial co-polymeric BETA (biphasic elastic thin for air−liquid interface cell culture conditions) scaffold, which were initially populated with human lung fibroblasts (IMR90 cell line), as the main generator of ECM proteins. Due to the interconnected porous structure of the thin (<5 µm) BETA scaffold, the cells can grow on and infiltrate into the scaffold and deposit their own ECM. After a mild decellularization procedure, the ECM proteins remained on the scaffold, which now closely mimicked the cellular microenvironment of pulmonary cells more realistically than the plain artificial scaffolds. We assessed several decellularization methods and found that 20 mM NH4OH and 0.1% Triton X100 with subsequent DNase treatment completely removed the fibroblasts (from the first cellularization) and maintains collagen I and IV as the key ECM proteins on the scaffold. We also showed the repopulation of the primary fibroblast from human (without chronic lung disease (non-CLD) donors) and human bronchial epithelial (16HBE14o−) cells on the ECM-integrated BETA scaffold. With this technique, we developed a biomimetic scaffold that can mimic both the physico-mechanical properties and the native microenvironment of the lung ECM. The results indicate the potential of the presented bioactive scaffold for LTE application.

Towards a gold standard functional readout to characterize In Vitro lung barriers.

The development of biomimetic in vitro lung models as an alternative to animal studies is urgent to improve the predictability of the pharmacokinetics of potential new drugs. For pharmacokinetics studies, advanced in vitro lung models such as lung-chips should mimic a functional air-blood barrier. Unlike in vivo conditions, stem/primary cells and cell lines do not necessarily form a functional and tight barrier when cultured in vitro. Here, we explore the two gold standard techniques for monitoring barrier integrity: transepithelial electrical resistance (TEER) and permeability. We discuss the advantages and limitations of these methods, provide recommendations for methodological improvements, and we elude on possible future directions.

Real-Time Measurement of Cell Mechanics as a Clinically Relevant Readout of an In Vitro Lung Fibrosis Model Established on a Bioinspired Basement Membrane.

Lung fibrosis, one of the major post-COVID complications, is a progressive and ultimately fatal disease without a cure. Here, an organ- and disease-specific in vitro mini-lung fibrosis model equipped with noninvasive real-time monitoring of cell mechanics is introduced as a functional readout. To establish an intricate multiculture model under physiologic conditions, a biomimetic ultrathin basement (biphasic elastic thin for air-liquid culture conditions, BETA) membrane (<1 µm) is developed with unique properties, including biocompatibility, permeability, and high elasticity (<10 kPa) for cell culturing under air-liquid interface and cyclic mechanical stretch conditions. The human-based triple coculture fibrosis model, which includes epithelial and endothelial cell lines combined with primary fibroblasts from idiopathic pulmonary fibrosis patients established on the BETA membrane, is integrated into a millifluidic bioreactor system (cyclic in vitro cell-stretch, CIVIC) with dose-controlled aerosolized drug delivery, mimicking inhalation therapy. The real-time measurement of cell/tissue stiffness (and compliance) is shown as a clinical biomarker of the progression/attenuation of fibrosis upon drug treatment, which is confirmed for inhaled Nintedanib-an antifibrosis drug. The mini-lung fibrosis model allows the combined longitudinal testing of pharmacodynamics and pharmacokinetics of drugs, which is expected to enhance the predictive capacity of preclinical models and hence facilitate the development of approved therapies for lung fibrosis.

An inter-laboratory effort to harmonize the cell-delivered in vitro dose of aerosolized materials.

Air-liquid interface (ALI) lung cell models cultured on permeable transwell inserts are increasingly used for respiratory hazard assessment requiring controlled aerosolization and deposition of any material on ALI cells. The approach presented herein aimed to assess the transwell insert-delivered dose of aerosolized materials using the VITROCELL® Cloud12 system, a commercially available aerosol-cell exposure system. An inter-laboratory comparison study was conducted with seven European partners having different levels of experience with the VITROCELL® Cloud12. A standard operating procedure (SOP) was developed and applied by all partners for aerosolized delivery of materials, i.e., a water-soluble molecular substance (fluorescence-spiked salt) and two poorly soluble particles, crystalline silica quartz (DQ12) and titanium dioxide nanoparticles (TiO2 NM-105). The material dose delivered to transwell inserts was quantified with spectrofluorometry (fluorescein) and with the quartz crystal microbalance (QCM) integrated in the VITROCELL® Cloud12 system. The shape and agglomeration state of the deposited particles were confirmed with transmission electron microscopy (TEM). Inter-laboratory comparison of the device-specific performance was conducted in two steps, first for molecular substances (fluorescein-spiked salt), and then for particles. Device- and/or handling-specific differences in aerosol deposition of VITROCELL® Cloud12 systems were characterized in terms of the so-called deposition factor (DF), which allows for prediction of the transwell insert-deposited particle dose from the particle concentration in the aerosolized suspension. Albeit DF varied between the different labs from 0.39 to 0.87 (mean (coefficient of variation (CV)): 0.64 (28%)), the QCM of each VITROCELL® Cloud 12 system accurately measured the respective transwell insert-deposited dose. Aerosolized delivery of DQ12 and TiO2 NM-105 particles showed good linearity (R2 > 0.95) between particle concentration of the aerosolized suspension and QCM-determined insert-delivered particle dose. The VITROCELL® Cloud 12 performance for DQ12 particles was identical to that for fluorescein-spiked salt, i.e., the ratio of measured and salt-predicted dose was 1.0 (29%). On the other hand, a ca. 2-fold reduced dose was observed for TiO2 NM-105 (0.54 (41%)), which was likely due to partial retention of TiO2 NM-105 agglomerates in the vibrating mesh nebulizer of the VITROCELL® Cloud12. This inter-laboratory comparison demonstrates that the QCM integrated in the VITROCELL® Cloud 12 is a reliable tool for dosimetry, which accounts for potential variations of the transwell insert-delivered dose due to device-, handling- and/or material-specific effects. With the detailed protocol presented herein, all seven partner laboratories were able to demonstrate dose-controlled aerosolization of material suspensions using the VITROCELL® Cloud12 exposure system at dose levels relevant for observing in vitro hazard responses. This is an important step towards regulatory approved implementation of ALI lung cell cultures for in vitro hazard assessment of aerosolized materials.

A drug screen with approved compounds identifies amlexanox as a novel Wnt/ß-catenin activator inducing lung epithelial organoid formation.

BACKGROUND AND PURPOSE: Emphysema is an incurable disease characterized by loss of lung tissue leading to impaired gas exchange. Wnt/ß-catenin signalling is reduced in emphysema, and exogenous activation of the pathway in experimental models in vivo and in human ex vivo lung tissue improves lung function and structure. We sought to identify a pharmaceutical able to activate Wnt/ß-catenin signalling and assess its potential to activate lung epithelial cells and repair. EXPERIMENTAL APPROACH: We screened 1216 human-approved compounds for Wnt/ß-catenin signalling activation using luciferase reporter cells and selected candidates based on their computationally predicted protein targets. We further performed confirmatory luciferase reporter and metabolic activity assays. Finally, we studied the regenerative potential in murine adult epithelial cell-derived lung organoids and in vivo using a murine elastase-induced emphysema model. KEY RESULTS: The primary screen identified 16 compounds that significantly induced Wnt/ß-catenin-dependent luciferase activity. Selected compounds activated Wnt/ß-catenin signalling without inducing cell toxicity or proliferation. Two compounds were able to promote organoid formation, which was reversed by pharmacological Wnt/ß-catenin inhibition, confirming the Wnt/ß-catenin-dependent mechanism of action. Amlexanox was used for in vivo evaluation, and preventive treatment resulted in improved lung function and structure in emphysematous mouse lungs. Moreover, gene expression of Hgf, an important alveolar repair marker, was increased, whereas disease marker Eln was decreased, indicating that amlexanox induces pro-regenerative signalling in emphysema. CONCLUSION AND IMPLICATIONS: Using a drug screen based on Wnt/ß-catenin activity, organoid assays and a murine emphysema model, amlexanox was identified as a novel potential therapeutic agent for emphysema.

A Bioinspired in vitro Lung Model to Study Particokinetics of Nano-/Microparticles Under Cyclic Stretch and Air-Liquid Interface Conditions.

Evolution has endowed the lung with exceptional design providing a large surface area for gas exchange area (ca. 100 m2) in a relatively small tissue volume (ca. 6 L). This is possible due to a complex tissue architecture that has resulted in one of the most challenging organs to be recreated in the lab. The need for realistic and robust in vitro lung models becomes even more evident as causal therapies, especially for chronic respiratory diseases, are lacking. Here, we describe the Cyclic I n VI tro Cell-stretch (CIVIC) "breathing" lung bioreactor for pulmonary epithelial cells at the air-liquid interface (ALI) experiencing cyclic stretch while monitoring stretch-related parameters (amplitude, frequency, and membrane elastic modulus) under real-time conditions. The previously described biomimetic copolymeric BETA membrane (5 µm thick, bioactive, porous, and elastic) was attempted to be improved for even more biomimetic permeability, elasticity (elastic modulus and stretchability), and bioactivity by changing its chemical composition. This biphasic membrane supports both the initial formation of a tight monolayer of pulmonary epithelial cells (A549 and 16HBE14o-) under submerged conditions and the subsequent cell-stretch experiments at the ALI without preconditioning of the membrane. The newly manufactured versions of the BETA membrane did not improve the characteristics of the previously determined optimum BETA membrane (9.35% PCL and 6.34% gelatin [w/v solvent]). Hence, the optimum BETA membrane was used to investigate quantitatively the role of physiologic cyclic mechanical stretch (10% linear stretch; 0.33 Hz: light exercise conditions) on size-dependent cellular uptake and transepithelial transport of nanoparticles (100 nm) and microparticles (1,000 nm) for alveolar epithelial cells (A549) under ALI conditions. Our results show that physiologic stretch enhances cellular uptake of 100 nm nanoparticles across the epithelial cell barrier, but the barrier becomes permeable for both nano- and micron-sized particles (100 and 1,000 nm). This suggests that currently used static in vitro assays may underestimate cellular uptake and transbarrier transport of nanoparticles in the lung.

Organ-restricted vascular delivery of nanoparticles for lung cancer therapy.

Nanoparticle-based targeted drug delivery holds promise for treatment of cancers. However, most approaches fail to be translated into clinical success due to ineffective tumor targeting in vivo. Here, the delivery potential of mesoporous silica nanoparticles (MSN) functionalized with targeting ligands for EGFR and CCR2 is explored in lung tumors. The addition of active targeting ligands on MSNs enhances their uptake in vitro but fails to promote specific delivery to tumors in vivo, when administered systemically via the blood or locally to the lung into immunocompetent murine lung cancer models. Ineffective tumor targeting is due to efficient clearance of the MSNs by the phagocytic cells of the liver, spleen, and lung. These limitations, however, are successfully overcome using a novel organ-restricted vascular delivery (ORVD) approach. ORVD in isolated and perfused mouse lungs of Kras-mutant mice enables effective nanoparticle extravasation from the tumor vasculature into the core of solid lung tumors. In this study, ORVD promotes tumor cell-specific uptake of nanoparticles at cellular resolution independent of their functionalization with targeting ligands. Organ-restricted vascular delivery thus opens new avenues for optimized nanoparticles for lung cancer therapy and may have broad applications for other vascularized tumor types.

The Preparation of Decellularized Mouse Lung Matrix Scaffolds for Analysis of Lung Regenerative Cell Potential.

Lung transplantation is the only option for patients with end-stage lung disease, but there is a shortage of available lung donors. Furthermore, efficiency of lung transplantation has been limited due to primary graft dysfunction. Recent mouse models mimicking lung disease in humans have allowed for deepening our understanding of disease pathomechanisms. Moreover, new techniques such as decellularization and recellularization have opened up new possibilities to contribute to our understanding of the regenerative mechanisms involved in the lung. Stripping the lung of its native cells allows for unprecedented analyses of extracellular matrix and sets a physiologic platform to study the regenerative potential of seeded cells. A comprehensive understanding of the molecular pathways involved for lung development and regeneration in mouse models can be translated to regeneration strategies in higher organisms, including humans. Here we describe and discuss several techniques used for murine lung de- and recellularization, methods for evaluation of efficacy including histology, protein/RNA isolation at the whole lung, as well as lung slices level.

Noninvasive Real-Time Assessment of Cell Viability in a Three-Dimensional Tissue.

Maintaining cell viability within 3D tissue engineering scaffolds is an essential step toward a functional tissue or organ. Assessment of cell viability in 3D scaffolds is necessary to control and optimize tissue culture process. Monitoring systems based on respiration activity of cells (e.g., oxygen consumption) have been used in various cell cultures. In this research, an online monitoring system based on respiration activity was developed to monitor cell viability within acellular lung scaffolds. First, acellular lung scaffolds were recellularized with human umbilical cord vein endothelial cells, and then, cell viability was monitored during a 5-day period. The real-time monitoring system generated a cell growth profile representing invaluable information on cell viability and proliferative states during the culture period. The cell growth profile obtained by the monitoring system was consistent with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis and glucose consumption measurement. This system provided a means for noninvasive, real-time, and repetitive investigation of cell viability. Also, we showed the applicability of this monitoring system by introducing shaking as an operating parameter in a long-term culture.

Advances in pulmonary therapy and drug development: Lung tissue engineering to lung-on-a-chip.

Lung disease is one of the major causes of death, and the rate of pulmonary diseases has been increasing for decades. Although lung transplantation is the only treatment for majority of patients, this method has been limited due to lack of donors. Therefore, recently, attentions have increased to some new strategies with the aid of tissue engineering and microfluidics techniques not only for the functional analysis, but also for drug screening. In fact, in tissue engineering, the engineered tissue is able to grow by using the patient's own cells without intervention in the immune system. On the other hand, microfluidics devices are applied in order to evaluate drug screenings, function analysis and toxicity. This article reviews new advances in lung tissue engineering and lung-on-a-chip. Furthermore, future directions, difficulties and drawbacks of pulmonary therapy in these areas are discussed.