Disease Models: Lung Models for Testing Drugs Against Inflammation and Infection.

Lung diseases have increasingly attracted interest in the past years. The all-known fear of failing treatments against severe pulmonary infections and plans of the pharmaceutical industry to limit research on anti-infectives to a minimum due to cost reasons makes infections of the lung nowadays a "hot topic." Inhalable antibiotics show promising efficacy while limiting adverse systemic effects to a minimum. Moreover, in times of increased life expectancy in developed countries, the treatment of chronic maladies implicating inflammatory diseases, like bronchial asthma or chronic obstructive pulmonary disease, becomes more and more exigent and still lacks proper treatment.In this chapter, we address in vitro models as well as necessary in vivo models to help develop new drugs for the treatment of various severe pulmonary diseases with a strong focus on infectious diseases. By first presenting the essential hands-on techniques for the setup of in vitro models, we intend to combine these with already successful and interesting model approaches to serve as some guideline for the development of future models. The overall goal is to maximize time and cost-efficacy and to minimize attrition as well as animal trials when developing novel anti-infective therapeutics.

Addressing the ADME Challenges of Compound Loss in a PDMS-Based Gut-on-Chip Microphysiological System.

Microphysiological systems (MPSs) are promising in vitro technologies for physiologically relevant predictions of the human absorption, distribution, metabolism, and excretion (ADME) properties of drug candidates. However, polydimethylsiloxane (PDMS), a common material used in MPSs, can both adsorb and absorb small molecules, thereby compromising experimental results. This study aimed to evaluate the feasibility of using the PDMS-based Emulate gut-on-chip to determine the first-pass intestinal drug clearance. In cell-free PDMS organ-chips, we assessed the loss of 17 drugs, among which testosterone was selected as a model compound for further study based on its substantial ad- and absorptions to organ chips and its extensive first-pass intestinal metabolism with well-characterized metabolites. A gut-on-chip model consisting of epithelial Caco-2 cells and primary human umbilical vein endothelial cells (HUVECs) was established. The barrier integrity of the model was tested with reference compounds and inhibition of drug efflux. Concentration-time profiles of testosterone were measured in cell-free organ chips and in gut-on-chip models. A method to deduce the metabolic clearance was provided. Our results demonstrate that metabolic clearance can be determined with PDMS-based MPSs despite substantial compound loss to the chip. Overall, this study offers a practical protocol to experimentally assess ADME properties in PDMS-based MPSs.

A Monoclonal Human Alveolar Epithelial Cell Line ("Arlo") with Pronounced Barrier Function for Studying Drug Permeability and Viral Infections.

In the development of orally inhaled drug products preclinical animal models regularly fail to predict pharmacological as well as toxicological responses in humans. Models based on human cells and tissues are potential alternatives to animal experimentation allowing for the isolation of essential processes of human biology and making them accessible in vitro. Here, the generation of a novel monoclonal cell line "Arlo," derived from the polyclonal human alveolar epithelium lentivirus immortalized cell line hAELVi via single-cell printing, and its characterization as a model for the human alveolar epithelium as well as a building block for future complex in vitro models is described. "Arlo" is systematically compared in vitro to primary human alveolar epithelial cells (hAEpCs) as well as to the polyclonal hAELVi cell line. "Arlo" cells show enhanced barrier properties with high transepithelial electrical resistance (TEER) of ≈3000 Ω cm2 and a potential difference (PD) of ≈30 mV under air-liquid interface (ALI) conditions, that can be modulated. The cells grow in a polarized monolayer and express genes relevant to barrier integrity as well as homeostasis as is observed in hAEpCs. Successful productive infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a proof-of-principle study offers an additional, attractive application of "Arlo" beyond biopharmaceutical experimentation.

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.

PerfuPul-A Versatile Perfusable Platform to Assess Permeability and Barrier Function of Air Exposed Pulmonary Epithelia.

Complex in vitro models, especially those based on human cells and tissues, may successfully reduce or even replace animal models within pre-clinical development of orally inhaled drug products. Microfluidic lung-on-chips are regarded as especially promising models since they allow the culture of lung specific cell types under physiological stimuli including perfusion and air-liquid interface (ALI) conditions within a precisely controlled in vitro environment. Currently, though, such models are not available to a broad user community given their need for sophisticated microfabrication techniques. They further require systematic comparison to well-based filter supports, in analogy to traditional Transwells®. We here present a versatile perfusable platform that combines the advantages of well-based filter supports with the benefits of perfusion, to assess barrier permeability of and aerosol deposition on ALI cultured pulmonary epithelial cells. The platform as well as the required technical accessories can be reproduced via a detailed step-by-step protocol and implemented in typical bio-/pharmaceutical laboratories without specific expertise in microfabrication methods nor the need to buy costly specialized equipment. Calu-3 cells cultured under liquid covered conditions (LCC) inside the platform showed similar development of transepithelial electrical resistance (TEER) over a period of 14 days as cells cultured on a traditional Transwell®. By using a customized deposition chamber, fluorescein sodium was nebulized via a clinically relevant Aerogen® Solo nebulizer onto Calu-3 cells cultured under ALI conditions within the platform. This not only allowed to analyze the transport of fluorescein sodium after ALI deposition under perfusion, but also to compare it to transport under traditional static conditions.

A Custom-Made Device for Reproducibly Depositing Pre-metered Doses of Nebulized Drugs on Pulmonary Cells in vitro.

The deposition of pre-metered doses (i.e., defined before and not after exposition) at the air-liquid interface of viable pulmonary epithelial cells remains an important but challenging task for developing aerosol medicines. While some devices allow quantification of the deposited dose after or during the experiment, e.g., gravimetrically, there is still no generally accepted way to deposit small pre-metered doses of aerosolized drugs or pharmaceutical formulations, e.g., nanomedicines. Here, we describe a straightforward custom-made device, allowing connection to commercially available nebulizers with standard cell culture plates. Designed to tightly fit into the approximately 12-mm opening of either a 12-well Transwell® insert or a single 24-well plate, a defined dose of an aerosolized liquid can be directly deposited precisely and reproducibly (4.8% deviation) at the air-liquid interface (ALI) of pulmonary cell cultures. The deposited dose can be controlled by the volume of the nebulized solution, which may vary in a range from 20 to 200 µl. The entire nebulization-deposition maneuver is completed after 30 s and is spatially homogenous. After phosphate-buffered saline (PBS) deposition, the viability and barrier properties transepithelial electrical resistance (TEER) of human bronchial epithelial Calu-3 cells were not negatively affected. Straightforward in manufacture and use, the device enables reproducible deposition of metered doses of aerosolized drugs to study the interactions with pulmonary cell cultures grown at ALI conditions.

Capturing the Onset of Bacterial Pulmonary Infection in Acini-On-Chips.

Bacterial invasion of the respiratory system leads to complex immune responses. In the deep alveolar regions, the first line of defense includes foremost the alveolar epithelium, the surfactant-rich liquid lining, and alveolar macrophages. Typical in vitro models come short of mimicking the complexity of the airway environment in the onset of airway infection; among others, they neither capture the relevant anatomical features nor the physiological flows innate of the acinar milieu. Here, novel microfluidic-based acini-on-chips that mimic more closely the native acinar airways at a true scale with an anatomically inspired, multigeneration alveolated tree are presented and an inhalation-like maneuver is delivered. Composed of human alveolar epithelial lentivirus immortalized cells and macrophages-like human THP-1 cells at an air-liquid interface, the models maintain critically an epithelial barrier with immune function. To demonstrate, the usability and versatility of the platforms, a realistic inhalation exposure assay mimicking bacterial infection is recapitulated, whereby the alveolar epithelium is exposed to lipopolysaccharides droplets directly aerosolized and the innate immune response is assessed by monitoring the secretion of IL8 cytokines. These efforts underscore the potential to deliver advanced in vitro biosystems that can provide new insights into drug screening as well as acute and subacute toxicity assays.