The Response of Human Nasal and Bronchial Organotypic Tissue Cultures to Repeated Whole Cigarette Smoke Exposure

Talikka, Marja; Kostadinova, Radina; Xiang, Yu‐Tao; Mathis, Carole; Sewer, Alain; Majeed, Shoaib; Kuehn, Diana; Frentzel, Stefan; Merg, Céline; Geertz, Marcel; Martin, Florian; Ivanov, Nikolai V.; Peitsch, Manuel C.; Hoeng, Julia

doi:10.1177/1091581814551647

Cited by 42 publications

(34 citation statements)

References 85 publications

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“…The air-exposed nature of these cultures makes them promising vehicles for toxicity studies of airborne substances (Iskandar et al 2015; Kogel et al 2015; Mathis et al 2013; Neilson et al 2015). Even chronic long-term or repeated exposures, to better mimic human occupational exposures, are realistic possibilities (Talikka et al 2014). Furthermore, use of cells from designated patient populations or exposure of the cells during differentiation to disease-specific factors, such as Th2 cytokines for allergic airways inflammation, can be used to generate disease-specific models.…”

Section: Introductionmentioning

confidence: 99%

Xenobiotic metabolism in differentiated human bronchial epithelial cells

et al. 2016

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Differentiated human bronchial epithelial cells in air liquid interface cultures (ALI-PBEC) represent a promising alternative for inhalation studies with rodents as these 3D airway epithelial tissue cultures recapitulate the human airway in multiple aspects, including morphology, cell type composition, gene expression and xenobiotic metabolism. We performed a detailed longitudinal gene expression analysis during the differentiation of submerged primary human bronchial epithelial cells into ALI-PBEC to assess the reproducibility and inter-individual variability of changes in transcriptional activity during this process. We generated ALI-PBEC cultures from four donors and focussed our analysis on the expression levels of 362 genes involved in biotransformation, which are of primary importance for toxicological studies. Expression of various of these genes (e.g., GSTA1, ADH1C, ALDH1A1, CYP2B6, CYP2F1, CYP4B1, CYP4X1 and CYP4Z1) was elevated following the mucociliary differentiation of airway epithelial cells into a pseudo-stratified epithelial layer. Although a substantial number of genes were differentially expressed between donors, the differences in fold changes were generally small. Metabolic activity measurements applying a variety of different cytochrome p450 substrates indicated that epithelial cultures at the early stages of differentiation are incapable of biotransformation. In contrast, mature ALI-PBEC cultures were proficient in the metabolic conversion of a variety of substrates albeit with considerable variation between donors. In summary, our data indicate a distinct increase in biotransformation capacity during differentiation of PBECs at the air–liquid interface and that the generation of biotransformation competent ALI-PBEC cultures is a reproducible process with little variability between cultures derived from four different donors.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-016-1868-7) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Xenobiotic metabolism in differentiated human bronchial epithelial cells

et al. 2016

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“…The recent development of 3-D organotypic nasal epithelial cell culture models, including the commercially available air-liquid interface human nasal culture MucilAir™, offer more physiologically relevant and robust systems for studying the effects of exposure through inhalation and for studying microbial infections on the nasal cavity. Air-liquid nasal cultures contain a fully differentiated, columnar pseudostratified epithelium consisting of basal cells, ciliated cells, and mucus producing goblet cells, the proportion of which resemble that of the in vivo nasal tissue (Constant et al, 2014;Talikka et al, 2014). These cultures respond to pro-inflammatory stimuli and the secretion of various chemokines, cytokines, and growth factors has been reported following exposure to cigarette smoke (CS) or rhinovirus infection (Yu et al, 2011).…”

Section: R4f Smoke and Ths22 Aerosol Exposure And Endpoint Generationmentioning

confidence: 99%

3-D nasal cultures: Systems toxicological assessment of a candidate modified-risk tobacco product

Iskandar

Mathis

Martin

et al. 2017

ALTEX

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ratory animals -to reduce the number of animals, to refine the design of procedures such that pain and distress are minimized, and to replace animal models with alternative methods and lower organisms when possible (Doke and Dhawale, 2015). In this regard, the capability to preserve human respiratory cells in culture provides a tool not only to generate mechanistic data, but also to improve the predicted effects of exposure hazard through inhalation in humans. An emphasis on assessment studies using human-derived in vitro culture systems will reduce the problems that are inherent to interspecies translatability (Manuppello and Sullivan, 2015).In vitro toxicology approaches have evolved from focusing on the molecular changes within a cell to understanding the toxicity-related mechanisms (cell-cell interactions) in models IntroductionMechanistic investigation of the impact of exposure on the lower airway remains challenging because access to lung tissues is limited. Although biopsy samples can be obtained from patients/donors for mechanistic studies of exposure-induced injury, a biopsy procedure is invasive and there are related ethical concerns (Peppercorn, 2013). Alternatively, animal studies allow the collection of biological samples from various animal models of human diseases. However, biological responses can vary among animal species, and findings obtained from animal studies may not apply to humans. Alternatives to animal testing have been proposed to overcome these drawbacks, including the 3Rs strategy -reduction, refinement, and replacement of labo- Research SummaryIn vitro toxicology approaches have evolved from a focus on molecular changes within a cell to understanding of toxicity-related mechanisms in systems that can mimic the in vivo environment. The recent development of three dimensional (3-D) organotypic nasal epithelial culture models offers a physiologically robust system for studying the effects of exposure through inhalation. Exposure to cigarette smoke (CS) is associated with nasal inflammation; thus, the nasal epithelium is relevant for evaluating the pathophysiological impact of CS exposure. The present study investigated further the application of in vitro human 3-D nasal epithelial culture models for toxicological assessment of inhalation exposure. Aligned with 3Rs strategy, this study aimed to explore the relevance of a human 3-D nasal culture model to assess the toxicological impact of aerosols generated from a candidate modified risk tobacco product (cMRTP), the Tobacco Heating System (THS) 2.2, as compared with smoke generated from reference cigarette 3R4F. A series of experimental repetitions, where multiple concentrations of THS2.2 aerosol and 3R4F smoke were applied, were conducted to obtain reproducible measurements to understand the cellular/molecular changes that occur following exposure. In agreement with "Toxicity Testing in the 21 st Century -a Vision and a Strategy", this study implemented a systems toxicology approach and found that for all tested concentrations the impa...

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“…The xenobiotic exposure such as drugs or pesticides modify cell transcriptome and produce molecules such as cytochrome P450s, CYP1A1 and CYP1B1 that can be detected, localized, and quantified using this technique. 3,20,31,41 RNAscope can be a useful tool in exploratory toxicology to characterize cell-specific responses to toxic stimuli within 3D-organotypic tissue models as part of a tiered approach. RNAscope may be used here as a follow-up analysis after conducting (global) gene expression analysis to further investigate mechanisms behind a biological impact.…”

Section: Discussionmentioning

confidence: 99%

“…The staining can be visualized under a standard brightfield microscope. [1][2][3][4] The RNAscope technology is based on hybridization of a pool of 20 specific ''Z'' probe pairs targeting 1000 bases of RNA while each pair hybridizes to an *50 base pair region of target RNA. The detection is carried out by specific binding of oligonucleotide preamplifier molecules linked to sev-eral amplifiers containing multiple chromogenic labels.…”

Section: Introductionmentioning

confidence: 99%

“…The preamplifier cannot bind to a single Z probe (nonpaired Z probe); the hybridization of three Z-pair probes is sufficient to obtain a detectable signal. Differently from standard RNA ISH, limited to detecting only highly expressed genes with low sensitivity and specificity, [2][3][4][5] this targetspecific probe design minimizes nonspecific off-target signals, thus resulting in highly specific staining As RNAscope technology virtually works for any gene from any species in any tissue, it has been applied on various biological samples in many fields, including colorectal tumors, 6 cerebellum in neuroscience, 7 lung tissue in developmental biology, 8 or liver in stem cell biology. 9 However, to date, RNAscope has not been used on three-dimensional (3D) organotypic cultures, one of the latest advances in cell culture technology.…”

Section: Introductionmentioning

confidence: 99%

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Optimization of a NovelIn SituHybridization Technology on 3D Organotypic Cell Cultures

NeauLaurent

LorinCharlotte

FrentzelStefan

et al. 2019

Applied In Vitro Toxicology

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Introduction: Developed by Advanced Cell Diagnostics, RNAscope Ò in situ hybridization technology enables detection of a target RNA in a cell-specific manner on formalin-fixed paraffin-embedded tissue sections and represents a good alternative to immunohistochemistry. The goal of this work is to illustrate an optimized protocol of the RNAscope technology to detect target genes in various human organotypic culture models (nasal, small airway, and gingival). These culture models retain the three-dimensional structure of native epithelium, mimic in vivo morphology and human physiology, and can be used as alternative sources to animal testing. Materials and Methods: After fixation and processing of five replicates of the three different organotypic cell cultures, the tissue morphology was checked by hematoxylin and eosin staining. The RNAscope protocols were optimized based on three crucial parameters: heat pretreatment, enzymatic digestion, and signal amplification. Digital images of the RNAscope stained slides were generated using the Hamamatsu NanoZoomer 2.0 slide scanner, and images were quantified using a custom-made plugin on Definiens Tissue Studio software (Definiens AG, Munich, Germany). Results: The tissue morphology demonstrates optimum fixation and processing for samples, while the optimized protocol for RNAscope shows preserved RNA with staining on the positive control probe with score ‡2 and no staining on the negative control probe with score <1. Discussion and Conclusion: RNAscope combined with organotypic cell cultures is a promising tool to better understand cell-specific RNA expression while implementing 3R (replace, reduce, and refine animal testing) principles.

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The Response of Human Nasal and Bronchial Organotypic Tissue Cultures to Repeated Whole Cigarette Smoke Exposure

Cited by 42 publications

References 85 publications

Xenobiotic metabolism in differentiated human bronchial epithelial cells

Xenobiotic metabolism in differentiated human bronchial epithelial cells

3-D nasal cultures: Systems toxicological assessment of a candidate modified-risk tobacco product

Optimization of a NovelIn SituHybridization Technology on 3D Organotypic Cell Cultures

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