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

Abstract

3D reconstituted respiratory epithelia have emerged as better in vitro models for toxicological testing compared to cell lines due to the conservation of key morphological features and functions. MucilAir™ is a commercially available human airway epithelia system that can potentially maintain functional attributes for up to a year, however, detailed mucociliary characteristics and xenobiotic metabolism relevant to inhaled pro-toxicant bioactivation is lacking. Here, we assessed in MucilAir™ some key biomarkers that are characteristic of the respiratory epithelia including morphology, function and xenobiotics metabolism. The end points that were measured included targeted proteomics using a panel of 243 airway surface liquid (ASL) proteins, cilia beat frequency (CBF), a qRT-PCR screen of xenobiotic metabolizing enzymes, and CYP2A6/13, CYP1A1/1B1 activity. Comparison of ASL proteomics with human sputum identified key proteins common to both matrices, but present at different levels. Xenobiotic metabolism gene profiling demonstrated strong similarities with the normal human lung and did not reveal any consistent changes when assessed over a 6 month period. Inducibility and activity of CYP1A1/1B1 and activity of CYP2A6/2A13 were present at one month in culture and maintained in one tested MucilAir™ donor for several months. In conclusion, MucilAir™ presented important morphological and metabolic characteristics of a mucociliary epithelium in short and long term culture. MucilAir™ is therefore a potentially useful model to test repeated sub-cytotoxic doses of toxicants.

Xenobiotic metabolism in differentiated human bronchial epithelial cells

“…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).…”

“…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.…”

“…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.…”

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

“…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).…”

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

“…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.…”