This study describes a complex human in vitro model for evaluating anti-inflammatory drug response in the alveoli that may contribute to the reduction of animal testing in the pre-clinical stage of drug development. The model is based on the human alveolar epithelial cell line Arlo co-cultured with macrophages differentiated from the THP-1 cell line, creating a physiological biological microenvironment. To mimic the three-dimensional architecture and dynamic expansion and relaxation of the air-blood-barrier, they are grown on a stretchable microphysiological lung-on-chip. For validating the in vitro model, three different protocols have been developed to demonstrate the clinically established anti-inflammatory effect of glucocorticoids to reduce certain inflammatory markers after different pro-inflammatory stimuli: (1) an inflammation caused by bacterial LPS (lipopolysaccharides) to simulate an LPS-induced acute lung injury measured best with cytokine IL-6 release; (2) an inflammation caused by LPS at ALI (air-liquid interface) to investigate aerosolized anti-inflammatory treatment, measured with chemokine IL-8 release; and (3) an inflammation with a combination of human inflammatory cytokines TNFα and IFNγ to simulate a critical cytokine storm leading to epithelial barrier disruption, where the eventual weakening or protection of the epithelial barrier can be measured. In all cases, the presence of macrophages appeared to be crucial to mediating inflammatory changes in the alveolar epithelium. LPS induction led to inflammatory changes independently of stretch conditions. Dynamic stretch, emulating breathing-like mechanics, was essential for in vitro modeling of the clinically relevant outcome of epithelial barrier disruption upon TNFα/IFNγ-induced inflammation.

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 cm 2 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.

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 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 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.

Serum concentrations of immunoglobulins G, A, M and IgG subclasses were determined by single radial immunodiffusion assay in a population of sickle cell anaemia patients resident in the tropics. Fifty apparently healthy subjects of haemoglobin genotype AA, of comparable age, sex and socioeconomic status (SES), and in the same environment as the patients, were included as controls. Three indices of morbidity in SCA, namely frequency of crisis, degree of anaemia and the number of organ complications, were used to derive a severity score for each patient; and thus categorize the subjects into severity groups. Immunoglobulin levels were then correlated with the indices of morbidity as well as the derived severity score. IgG, IgA, IgM, IgG1 and IgG3 levels were significantly raised in the SCA subjects when they were compared as a group with the controls. When separated into disease severity groups, the mildly affected patients were found to have virtually normal levels of immunoglobulins. Total IgG concentration and level of the IgG3 subclass showed significant positive correlation with frequency of crisis and derived severity score. Markedly raised levels of IgG and IgG3 may be predictive of severity in sickle cell anaemia.

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.

The interplay of particles with pulmonary surfactant, a lipid‐protein material pivotal for lung function, is hypothesized as a key factor that has not been routinely considered in the current in vitro models when determining the fate of inhaled nanomaterials. To explain its influence on cellular uptake and protective effects, nanoparticles are studied on two models of alveolar cells, in the absence or presence of pulmonary surfactant. Composition and interfacial performance of native human and porcine surfactants, a commercially available bovine surfactant (Alveofact), and an artificial lung lining fluid are characterized using shotgun lipidomics and biophysical approaches (i.e., Langmuir surface balances and captive bubble surfactometry). Plain and aminofunctionalized silica nanoparticles and a novel antimycobacterial nanoformulated benzothiazinone (BTZ043) are selected as examples of neutral, positively charged and therapeutically relevant nanoparticles, respectively. They are deposited onto monocultures of human alveolar epithelial and phagocytic cell lines in the presence or absence of the surfactant preparations, modeling the alveolar milieu. Only surfactant preparations with high interfacial activity and distinctive composition mitigated the toxicity of aerosolized particles, along with a tendency of aerosolized particles to aggregate. Key requirements of surfactant preparations needed when studying interactions of nanomaterials with the pulmonary air‐blood barrier in vitro are identified.

The pharmaceutical industry is in rising need for alternative methods that minimise animal testing during early drug development, mainly because of the poor transferability from animal to human, costs or ethical issues. However, traditional cellular models fail to reproduce essential physio-pathological, systemic and structural aspects. In this sense, organ-on-chips have emerged as advanced in vitro systems emulating organ microenvironment. Due to the complex architecture and dynamics of the lungs, a significant way of entry for pathogens and pharmaceutical agents, we aim to develop a robust and versatile breathing alveolar model on chip to mimic better the physio-pathological milieu. We used the new AX12 setup (based on Stucki et al. Scientific Reports 2018, 33:14359), which enables physiological stretch and aerosol exposure, to model (1) alveolar inflammation and (2) anti-inflammatory drug administration. To do so, we generated a human alveolar barrier by co-culturing alveolar epithelial cells (hAELVi; Kuehn et al. Altex 2016, 33(3):251-60) with monocyte-derived macrophages (THP-1). To induce inflammation, cells were incubated with lipopolysaccharide (LPS) during 24h in static and breathing conditions. After 6h of LPS initial exposure, the anti-inflammatory drug budesonide was administered. Cytokine release was analysed by FACS, showing an increase in proinflammatory cytokines upon LPS exposure, and TEER and confocal imaging were used to follow the integrity of the epithelial barrier. Promising results were obtained highlighting the potential of this model for emulating respiratory lung diseases and aerosol therapy.

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.

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.

2D in vitro lung cell models are useful tools to investigate safety and efficacy of aerosols. In order to better analyse complex connections in vivo, these models help to study various questions of cell-cell communication, inflammation, damage via external noxae, pathogenesis of diseases, and treatment with active pharmaceutical ingredients. This chapter illustrates the initiation of cell-based 2D in vitro models and limitations of in vivo situations to point out necessary properties. These comprise amongst others stable barrier properties and physiological qualities such as air-liquid interface and stretching properties. To model the lung epithelium, selection of a suited cell line is fundamental. Therefore, various cell types are exemplified with respect to their origin (primary cells versus cell lines) and to their physiological location (bronchial versus alveolar cells). Subsequently, co-cultures of different cell types are addressed and validated. To test these models, aerosol deposition is a key aspect, as it mimics the inhalation process in vivo. By summing up conventional and innovative devices, important elements of deposition on cell cultures are explained as it comes to test safety and efficacy of aerosols and inhalable formulations.