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In Vitro Systems and Inhalation Toxicology

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In early July, experts from around the world met in Nice, France, to share and discuss the latest innovations and research in the context of human lung in vitro models. The Lung In Vitro Event 2018 (LIVe) brought together scientists from academia, regulatory agencies, and different industries such as pharmaceuticals, biotechnology, tobacco, consumer goods, and medical devices. Topics covered included the applications of in vitro lung models for the investigation of infectious diseases, respiratory allergies, inhalation toxicology, and the development of biomedicals for conditions including chronic obstructive pulmonary disease (COPD), asthma, idiopathic pulmonary fibrosis (IPF), and lung cancer.

Philip Morris International (PMI) had a significant presence at LIVe, where a team of scientists and laboratory technicians were present to share scientific findings and expertise. As part of the pre-clinical assessment of Reduced-Risk Products (RRPs)*, PMI has developed and utilized advanced three-dimensional, organotypic systems derived from epithelial cells isolated from lung, mouth, and nasal cavities of healthy human donors. These systems have been used in several in vitro studies, details of which were presented at LIVe.1 Further studies presented at the conference investigated:

  • The potential of High-Content Imaging for the evaluation of complex in vitro cellular systems2
  • A series of non-invasive approaches commonly used to characterize the three-dimensional lung model MucilAir™ 3,4
  • An innovative microfluidic organ-on-a-chip model combining both lung and liver tissues5

The studies and innovations were demonstrated not only to have applications related to the assessment of RRPs, but also for pharmaceutical drug discovery, environmental health studies, and broader toxicological research. In addition, they all also support the vision of the widely accepted 3Rs principles for animal testing: the replacement, reduction and refinement of animal use in scientific research.

Three-Dimensional Models: Traditional Endpoints Combined with Cellular Systems Toxicology

The generation of RRP aerosols has previously been proved to result in a greatly reduced formation of the harmful and potentially harmful constituents (HPHCs) commonly found in cigarette smoke (of at least 90% for one of our RRPs). Our three-dimensional organotypic in vitro models were used in proof-of-concept studies to assess whether the reduction in exposure to HPHCs translates into a reduced biological impact at the cellular level. This was made possible by the configuration of the three-dimensional organotypic cultures, in which cells are maintained at the air-liquid interface and thus enable direct exposure to smoke and aerosols.

Following the exposure of lung, oral, and nasal epithelial cell cultures to either cigarette smoke or RRP aerosol, biological effects were assessed at various time-points and using an array of endpoints. These included well-established in vitro testing endpoints such as deposition of toxic carbonyls, morphological alterations, and secretion of inflammatory mediators, as well as systems toxicological measurements involving transcriptomics, proteomics, and metabolomics. Collected data were also processed through a set of manually curated biological network models that are known to relate to respiratory disease. Biological impacts were then quantified, compared, and complemented by standard gene-set analysis.

Multiple experimental repetitions ensured the robustness, reliability and reproducibility of data. The assessment showed that similar biological networks are perturbed following exposure to RRP aerosols as those perturbed following exposure to cigarette smoke, though at a much lesser magnitude and showing only a transient effect. In addition, RRP aerosols did not show any impact on other biological networks, distinct from those impacted by cigarette smoke.

High Content Imaging

High Content Imaging (HCI) is a well-established technique for the analysis of cell cultures grown in two dimensions. However, the development of three-dimensional systems has added significant complexity to the HCI process for obvious reasons. To evaluate the potential of HCI in these more complex in vitro systems, we developed a three-dimensional model of human bronchial epithelial cells in which we induced increased proliferation of goblet cells (one cell type commonly found in the respiratory tract) through the dosing of interleukin-13 (IL-13). The resulting effects were then examined through targeted antibody staining and image-based analysis.

The approach demonstrated that HCI can be used to evaluate complex, three-dimensional in vitro models. An evaluation of the expression and distribution of a marker related to goblet cells (Muc5AC) was used to identify and quantify IL-13 induced increase in goblet cell density, demonstrating the ability of our HCI methodology to accurately assess specific phenotypic changes upon treatment. In addition, the HCI was able to quickly and accurately identify structural damage and tissue abnormalities, and as such can be also be exploited as a tool for quality control of tissues.

MucilAir™ for Inhalation Toxicology

Among commercially available airway epithelial tissues, MucilAir™ has become a reference among in vitro options for respiratory toxicology. In partnership with British American Tobacco (BAT) and Epithelix (the producer of MucilAir™), we examined a series of parameters that are commonly used to characterize the MucilAir™ model. The intention was to accurately and comprehensively determine the suitability of MucilAir™ for the specific requirements of in vitro inhalation toxicology. The study was carried out independently in three separate laboratories and using standardized methodologies. Three different donors and six independent preparations of airway epithelial tissues were used to perform the study. The results, together with earlier findings on tissue stability, demonstrated the suitability of MucilAir™ as a test system for in vitro inhalation toxicity. However, batch-to-batch and donor-specific variations need to be taken into account when considering multisite comparisons.

The Lung-Liver-on-a-Chip

Three-dimensional in vitro models can be further enhanced through their incorporation into small devices that reproduce the complex microenvironments of specific organs. These are often referred to as ‘organs-on-chips’. While they typically only represent a single organ, and therefore cannot be used to study the organ-to-organ interactions observed in the human body, PMI has developed a combined lung-liver-on-a-chip. This is a fundamentally important development because while airborne compounds (such as cigarette smoke or RRP aerosols) are absorbed by the lung, their true toxicity may only be fully observed following their metabolization by the liver. At LIVe, we presented the methodology and results of a study which demonstrated the ability of our lung-liver-on-a-chip to hold both lung and liver tissues in a stable state for at least four weeks. In addition, the study demonstrated the metabolic capacity of the model and thus its potential for in vitro inhalation toxicology.

Summary and Conclusions

LIVe brought together international experts on in vitro models of the human lung. Available models are showing important progress in the effort to mimic human biology by increasing cell culture complexity, and the potential of these models to replace the use of animals in scientific research is clearly apparent. The conference also highlighted the significant value of collaboration, showing what can be achieved when academia, regulatory bodies, start-ups and industry work together. Leading pharmaceutical companies showed interest in the gene expression data sets generated from our three-dimensional models as they may be a potential resource for the investigation of immunosuppression in smokers with COPD. In addition, interest was expressed in our HCI methods as they could be exploited for the drug discovery and development process. 

In vitro models and related innovations are showing huge potential across a range of disciplines and study types. PMI looks forward to contributing to further developments in the field. 

Diego Marescotti is the High Content Screening Manager in the System Toxicology department of Philip Morris International R&D. After completing his PhD in Oncology and Molecular Pharmacology at the University of Ferrara (Italy) he continued his research investigating the mechanism of tumor escape from immune surveillance. He joined PMI in 2012 and is now leading a team of researchers which operates a high-throughput system for compound profiling.

1. Frentzel, S. In vitro assessment of reduced risk products (RRPs) using 3D-organotypic air-liquid interface cultures (MucilAir™ and SmallAir™) and the Vitrocell® 24/48 system. Lung In Vitro Event, Nice, France, July 5-6, 2018.
2. Marescotti, D. et al. High-content screening-based phenotypic analysis of organotypic 3D bronchial tissues. Lung In Vitro Event, Nice, France, July 5-6, 2018.
3. Constant, S. et al. Inter-laboratory performance of mucin ELLA, CBF, and TEER measurements on bronchial MucilAir™ cultures. Lung In Vitro Event, Nice, France, July 5-6, 2018.
4. Ortega Torres, L. et al. An assay to characterize the impact of cigarette smoke exposure on mucociliary clearance in vitro. Lung In Vitro Event, Nice, France, July 5-6, 2018.
5. Bovard, D. et al. Characterization of a lung/liver organ-on-a-chip model. Lung In Vitro Event, Nice, France, July 5-6, 2018.