Targeted omics analyses, and metabolic enzyme activity assays demonstrate maintenance of key mucociliary characteristics in long term cultures of reconstituted human airway epithelia

Baxter, Andrew; Thain, Simon C.; Banerjee, Anisha; Haswell, Linsey E.; Parmar, Aleesha; Phillips, Gary; Minet, Emmanuel

doi:10.1016/j.tiv.2015.03.004

Cited by 52 publications

(41 citation statements)

References 39 publications

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“…As a control, we investigated the transcriptional response of ALI-PBEC to TCDD, the prototypical aryl hydrocarbon receptor (AhR) ligand. Gene expression analysis of cultures 24 and 72 h after the start of exposure showed for all three compounds a strong induction of CYP1A1 as reported previously (Baxter et al 2015; Newland et al 2011), while the high basal expression of CYP1B1 was further up-regulated (Fig. 3).…”

Section: Resultssupporting

confidence: 85%

“…ALI-PBEC can be brought into continuous dynamic contact with chemicals in a test atmosphere simulating in vivo inhalation exposure. Gene expression studies have shown that these 3D respiratory epithelial cultures closely resemble in vivo airway epithelia (Dvorak et al 2011; Pezzulo et al 2011), while also the enzymatic activity of various CYPs such as CYP1A1/1B1 and CYP2A6/2A13 is conserved (Baxter et al 2015; Newland et al 2011). In the present study, we determined the reproducibility and inter-individual variation of changes in gene activity of genes involved in metabolism during the differentiation of primary human bronchial epithelial cells into mature ALI-PBEC.…”

Section: Discussionmentioning

confidence: 99%

“…For these purposes, it is important that these ALI cultures mimic human lung epithelium in their ability to biotransform xenobiotic chemicals which is supported by the presence of differentiated cell types such as club cells that have been implicated in biotransformation processes in the lung (Chichester et al 1991; Hukkanen et al 2002). Transcriptome analyses showed that these organotypic cultures recapitulate the transcriptional profile of in vivo airway epithelia (Dvorak et al 2011; Pezzulo et al 2011) while metabolic activity of for instance CYP1A1/1B1 and CYP2A6/2A13 is conserved (Baxter et al 2015; Newland et al 2011). In this study, we aimed to get a better insight into the temporal changes in gene activity during the differentiation of submerged primary human bronchial epithelial cells into ALI-PBEC and to assess the reproducibility and inter-individual variability of changes in transcriptional activity during this process.…”

Section: Introductionmentioning

confidence: 99%

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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: Resultssupporting

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Xenobiotic metabolism in differentiated human bronchial epithelial cells

et al. 2016

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“…Indeed not only morphological studies, but also gene expression studies have shown that ALI cultures recapitulate the gene expression pattern of airway epithelial cells in situ (Pezzulo et al, 2011;Dvorak et al, 2011). Furthermore, such ALI-differentiated cultures do maintain some of the key metabolic enzymes (Baxter et al, 2015). We recently showed that the ALI-differentiated cultures used in our laboratory for exposures to e.g.…”

Section: Primary Lung Epithelial Cellsmentioning

confidence: 87%

Human lung epithelial cell cultures for analysis of inhaled toxicants: Lessons learned and future directions

Hiemstra

Grootaers

Does

et al. 2018

Toxicology in Vitro

153

116

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A B S T R A C TThe epithelium that covers the conducting airways and alveoli is a primary target for inhaled toxic substances, and therefore a focus in inhalation toxicology. The increasing concern about the use of animal models has stimulated the development of in vitro cell culture models for analysis of the biological effects of inhaled toxicants. However, the validity of the current in vitro models and their acceptance by regulatory authorities as an alternative to animal models is a reason for concern, and requires a critical review. In this review, focused on human lung epithelial cell cultures as a model for inhalation toxicology, we discuss the choice of cells for these models, the cell culture system used, the method of exposure as well as the various read-outs to assess the cellular response. We argue that rapid developments in the 3D culture of primary epithelial cells, the use of induced pluripotent stem cells for generation of lung epithelial cells and the development of organ-on-a-chip technology are among the important developments that will allow significant advances in this field. Furthermore, we discuss the various routes of application of inhaled toxicants by air-liquid interface models as well as the vast array of read-outs that may provide essential information. We conclude that close collaboration between researchers from various disciplines is essential for development of valid methods that are suitable for replacement of animal studies for inhalation toxicology.

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“…For viral infections, MucilAir™-Pool airway epithelia were reconstituted with a mixture of human nasal cells isolated from 14 different donors, and cultured at the air-liquid interface (ALI) in MucilAir™ culture medium (EP04MM), ready-to-use, chemically defined, serum-free (Epithelix Sàrl, Geneva, Switzerland), in 24-well plates with 6.5-mm Transwell ® inserts (cat #3470, Corning Incorporated, Tweksbury, USA). The MucilAir™ models are stable for months and generate reproducible data in several domains, such as toxicology, therapeutic drug study or disease model (Baxter et al, 2015), (Balogh Sivars et al, 2017), (Rocca et al, 2016), (Sonneville et al, 2017).…”

Section: Mucilair™ Culturingmentioning

confidence: 99%

Antiviral drug screening by assessing epithelial functions and innate immune responses in human 3D airway epithelium model

et al. 2018

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Respiratory viral infections cause mild to severe diseases, such as common cold, bronchiolitis and pneumonia and are associated with substantial burden for society. To test new molecules for shortening, alleviating the diseases or to develop new therapies, relevant human in vitro models are mandatory. MucilAir™, a human standardized air-liquid interface 3D airway epithelial culture holds in vitro specific mechanisms to counter invaders comparable to the in vivo situation, such as mucus production, mucociliary clearance, and secretion of defensive molecules. The objective of this study was to test the relevance of such a model for the discovery and validation of antiviral drugs. Fully differentiated 3D nasal epithelium cultures were inoculated with picornaviruses, a coronavirus and influenza A viruses in the absence or in the presence of reference antiviral drugs. Results showed that, rupintrivir efficiently inhibits the replication of respiratory picornaviruses in a dose dependent manner and prevents the impairment of the mucociliary clearance. Similarly, oseltamivir reduced the replication of influenza A viruses in a dose dependent manner and prevented the impairment of the epithelial barrier function and cytotoxicity until 4 days of infection. In addition we found that Rhinovirus B14, C15 and influenza A(H1N1) induce significant increase of β Defensins 2 and Cathelicidin release with different time course. These results reveal that a large panel of epithelial functions is modified upon viral infection and validate MucilAir™ as a pertinent tool for pre-clinical antiviral drug testing.

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Targeted omics analyses, and metabolic enzyme activity assays demonstrate maintenance of key mucociliary characteristics in long term cultures of reconstituted human airway epithelia

Cited by 52 publications

References 39 publications

Xenobiotic metabolism in differentiated human bronchial epithelial cells

Xenobiotic metabolism in differentiated human bronchial epithelial cells

Human lung epithelial cell cultures for analysis of inhaled toxicants: Lessons learned and future directions

Antiviral drug screening by assessing epithelial functions and innate immune responses in human 3D airway epithelium model

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