In accordance with the well-accepted 3R strategy (the effort to “replace, reduce, refine” the use of animal models in research2), we have devised a series of human in vitro models which will avoid species translatability issues. These models are composed of human primary cells, cultured in two- or three-dimensions, which can be exposed to individual smoke constituents and fractions (eg, 2D cultures such as normal human bronchial epithelial cells or human endothelial cells) or to whole aerosols from Reduced-Risk Products (RRPs) or cigarette smoke (eg, 3D cultures).
By using systems toxicological risk assessment approaches, combining computable biological network models and gene expression changes, the molecular alterations triggered after different exposure conditions are analyzed and integrated with other biological endpoints. The measured biological alterations are also quantified, allowing the direct comparison of the impact triggered by exposure to cigarette smoke or RRP aerosols, thus providing evidence on the potential risk reduction.
Human 2D models and High Content Screening
High Content Screening (HCS) is an automated technology used to carry out functional / biological assays in a rapid, robust and cost-effective manner. The technology is based on the integration of three elements:
A state-of the art fluorescence microscope that allows the visualisation of cells and cellular structures.
A high-throughput image acquisition system that allows the automatic acquisition of hundreds of microscope images in a rapid and reproducible manner.
A software tool that enables the analysis and quantification of the biological signals in each one of the acquired images.
Using a combination of antibodies (capable of identifying specific components in the cell) and fluorescent dyes, the HCS technology can detect changes in target intensity, localisation and cellular morphology in live or fixed cell3.
At PMI, we use a large battery of HCS assays to investigate how specific substances alter the phenotype of a cell in a particular manner. Currently, we have established more than 10 different assays, allowing us to measure more than 15 different cellular parameters in cellular types representing the lung (normal human bronchial epithelial cells) and the cardiovascular system (human coronary artery endothelial cells)4.
Human 3D models
While 2D models have their uses, traditional 2D cell cultures in plastic dishes or flasks are limited in that the culture medium that covers them does not allow for direct exposure to complex aerosols. In addition, 2D cell cultures do not satisfactorily recapitulate the physiological cell-cell interaction conditions that cells experience in vivo.
To address these issues, in recent years researchers have looked for ways to culture cells in a more physiologically relevant manner. It has been demonstrated that by culturing normal human bronchial epithelial cells at the air-liquid interface, they are able to differentiate into a bronchial epithelium-like tissue with all the morphological properties that define the bronchial epithelium in vivo5. A 3D airway cellular model has also been built where cells express a comparable gene expression profile to tracheal and bronchial epithelial cells that have been collected in the clinical setting by brushing human airways6.
Cell culture in 3D has been touted as ‘biology's new dimension’7 and the National Institutes of Health, the US Food and Drug Administration, the Defense Advanced Research Projects Agency and the Defense Threat Reduction Agency are sponsoring a variety of projects focused on developing 3D organ models8.
At PMI we are using human 3D cultures that represent the different airway epithelia (nasal, oral, gingival and bronchial) that are directly exposed to cigarette smoke when inhaled by a smoker. These models are cultured with an air-liquid interface, allowing us to test whole cigarette smoke and RRP aerosols using the VITROCELL® system. We recently demonstrated that nasal, bronchial and oral in vitro systems are reliable models to assess RRPs by comparing their response to cigarette smoke exposure against in vivo tissue counterparts9,10,11,12.
 National Research Council Committee on Toxicity Testing Assessment of Environmental Agents. Toxicity testing in the 21st century: a vision and a strategy. 2007: National Academies Press.
 Russell, WMS, RL Burch, and CW Hume. The principles of humane experimental technique. 1959.
 Thermo Scientific. CellInsight NXT High Content Screening Platform Brochure. 2012.
 Gonzalez-Suarez, I, et al. Systems biology approach for evaluating the biological impact of environmental toxicants in vitro. Chem Res Toxicol, 2014. 27(3): p. 367-76.
 Karp, PH, et al. An in vitro model of differentiated human airway epithelia. Methods for establishing primary cultures. Methods Mol Biol, 2002. 188: p. 115-37.
 Pezzulo, AA, et al. The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia. Am J Physiol Lung Cell Mol Physiol, 2011. 300(1): p. L25-31.
 Abbott, A. Cell culture: Biology's new dimension. 2014.
 Baker, M. Tissue models: a living system on a chip. Nature, 2011. 471(7340): p. 661-5.
 Mathis, C, et al Human bronchial epithelial cells exposed in vitro to cigarette smoke at the air-liquid interface resemble bronchial epithelium from human smokers. Am J Physiol Lung Cell Mol Physiol, 2013. 304(7): p. L489-503.
 Iskandar, AR, et al. Systems approaches evaluating the perturbation of xenobiotic metabolism in response to cigarette smoke exposure in nasal and bronchial tissues. Biomed Res Int, 2013. 2013: p. 512086.
 Talikka, M, et al. The response of human nasal and bronchial organotypic tissue cultures to repeated whole cigarette smoke exposure. Int J Toxicol, 2014.
 Schlage, WK, et al. In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures. Toxicol Mech Methods, 2014. 24(7): p. 470-87.