bronchial epithelium

/Tag:bronchial epithelium

A Normal and Biotransforming Model of the Human Bronchial Epithelium for the Toxicity Testing of Aerosols and Solubilised Substances

Zoë C. Prytherch and Kelly A. BéruBé

In this article, we provide an overview of the experimental workflow by the Lung and Particle Research Group at Cardiff University, that led to the development of the two in vitro lung models — the normal human bronchial epithelium (NHBE) model and the lung–liver model, Metabo-Lung™. This work was jointly awarded the 2013 Lush Science Prize. The NHBE model is a three-dimensional, in vitro, human tissue-based model of the normal human bronchial epithelium, and Metabo-Lung involves the co-culture of the NHBE model with primary human hepatocytes, thus permitting the biotransformation of inhaled toxicants in an in vivo-like manner. Both models can be used as alternative test systems that could replace the use of animals in research and development for safety and toxicity testing in a variety of industries (e.g. the pharmaceutical, environmental, cosmetics, and food industries). Metabo-Lung itself is a unique tool for the in vitro detection of toxins produced by reactive metabolites. This 21st century animal replacement model could yield representative in vitro predictions for in vivo toxicity. This advancement in in vitro toxicology relies on filter-well technology that will enable a wide-spectrum of researchers to create viable and economic alternatives for respiratory safety assessment and disease-focused research.
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A Review of In Vitro Modelling Approaches to the Identification and Modulation of Squamous Metaplasia in the Human Tracheobronchial Epithelium

Alison C. Gray, Julie D. McLeod and Richard H. Clothier

Squamous metaplasia in the tracheobronchial epithelium (TBE) involves the replacement of the normal pseudostratified mucociliary epithelium with a stratified squamous epithelium. Squamous metaplasia is considered to be an adaptive response that protects the lumen from the effects of inhaled airborne pollutants, but which might also feature as a pre-neoplastic lesion preceding squamous cell carcinoma. With the exception of transglutaminase I, involucrin, and cytokeratins 5, 6 and 13, few markers that contribute to the squamous phenotype have been identified in human TBE that can be used in diagnosis or to monitor its development in laboratory investigations, and current models are inadequate to provide statistically meaningful data. Therefore, new predictive markers have been identified, and new techniques established, in epithelial in vitro models capable of expressing squamous characteristics, which will be used to identify hazardous exposures and elucidate the mechanisms by which they induce their effects. A protocol for the quantitative detection of transglutaminase activity has been standardised in keratinocytes, based on the enzymatic incorporation of fluorescein–cadaverine (FC) into bis(γ-glutamyl) polyamine cross-links. The specificity of this compound as a transglutaminase substrate was demonstrated by using a range of competitive transglutaminase inhibitors, and by modulation of the squamous pathway. FC incorporation was localised to the cell membrane of terminally differentiating cells, and was not visible in basal, proliferating cells. High calcium-containing medium, nicotine and cigarette smoke condensates (CSC) induced an increase in FC incorporation, providing evidence of their role in enhancing the squamous pathway. Analysis by flow cytometry was used to provide a quantitative assessment of a range of optimised squamous differentiation markers, identified in normal human bronchial epithelia and in a bronchial cell line. Transglutaminase I was induced in a time-dependent manner, in post-confluent cells induced to differentiate down the squamous pathway, whereas involucrin was ubiquitously expressed and the levels of cytokeratins 5, 6 and 18 were reduced. The response of these and other differentiation markers to squamous-inducing conditions is being explored.
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Filter-well Technology for Advanced Three-dimensional Cell Culture: Perspectives for Respiratory Research

Kelly BéruBé, Aldo Pitt, Patrick Hayden, Zoë Prytherch and Claire Job

Cell culture has long been a valuable tool for studying cell behaviour. Classical plastic substrates are two-dimensional, and usually promote cellular proliferation and inhibit differentiation. Understanding cell behaviour within complex multicellular tissues requires the systematic study of cells within the context of specific model microenvironments. A model system must mimic, to a certain degree, the in vivo situation, but, at the same time, can significantly reduce its complexity. There is increasing agreement that moving up to the third dimension provides a more physiologically-relevant and predictive model system. Moreover, many cellular processes (morphogenesis, organogenesis and pathogenesis) have been confirmed to occur exclusively when cells are ordered in a three-dimensional (3- D) manner. In order to achieve the desired in vivo phenotype, researchers can use microporous membranes for improved in vitro cell culture experiments. In the present review, we discuss the applications of filter-well technology for the advanced 3-D cell culture of human pulmonary cells.
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Medical Waste Tissues — Breathing Life back into Respiratory Research

Kelly A. BéruBé

With the advent of biobanks to store human lung cells and tissues from patient donations and from the procurement of medical waste tissues, it is now possible to integrate (both spatially and temporally) cells into anatomically-correct and physiologically-functional tissues. Modern inhalation toxicology relies on human data on exposure and adverse effects, to determine the most appropriate risk assessments and mitigations for beneficial respiratory health. A point in case is the recapitulation of airway tissue, such as the bronchial epithelium, to investigate the impact of air pollution on human respiratory health. The bronchi are the first point of contact for inhaled substances that bypass defences in the upper respiratory tract. Animal models have been used to resolve such inhalation toxicology hazards. However, the access to medical waste tissues has enabled the Lung Particle Research Group to tissue-engineer the Micro-Lung™ and Metabo-Lung™ cell culture models, as alternatives to animals in basic research and in the safety testing of aerosolised consumer goods. The former model favours investigations focused on lung injury and repair mechanisms, and the latter model provides the element of metabolism, through the co-culturing of lung and liver (hepatocyte) cells. These innovations represent examples of the animal-free alternatives advocated by the 21st century toxicology paradigm, whereby human-derived cell/tissue data will lead to more accurate and more-reliable public health risk assessments and therapeutic mitigations (e.g. exposure to ambient air pollutants and adverse drug reactions) for lung disease.
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