Validation of in vitro models for smoke exposure of primary human bronchial epithelial cells

Abstract

Rationale. The bronchial epithelium is constantly challenged by inhalative insults including cigarette smoke (CS), a key risk factor for lung disease. In vitro exposure of bronchial epithelial cells using CS extract (CSE) is a widespread alternative to whole CS (wCS) exposure. However, CSE exposure protocols vary considerably between studies, precluding direct comparison of applied doses. Moreover, they are rarely validated in terms of physiological response in vivo and the relevance of the findings is often unclear. Methods. We tested six different exposure settings in primary human bronchial epithelial cells (phBECs), including five CSE protocols in comparison with wCS exposure. We quantified cell-delivered dose and directly compared all exposures using expression analysis of 10 well-established smoke-induced genes in bronchial epithelial cells. CSE exposure of phBECs was varied in terms of differentiation state, exposure route, duration of exposure, and dose. Gene expression was assessed by quantitative Real-Time PCR (qPCR) and Western Blot analysis. Cell type-specific expression of smoke-induced genes was analyzed by immunofluorescent analysis. Results. Three surprisingly dissimilar exposure types, namely chronic CSE treatment of differentiating phBECs, acute CSE treatment of submerged basal phBECs, and wCS exposure of differentiated phBECs performed best, resulting in significant upregulation of seven (chronic CSE) and six (acute wCS, acute submerged CSE exposure) out of 10 genes. Acute apical or basolateral exposure of differentiated phBECs with CSE was much less effective despite similar doses used. Conclusions. Our findings provide guidance for the design of human in vitro CS exposure models in experimental and translational lung research.

Quantitative proteomics of differentiated primary bronchial epithelial cells from chronic obstructive pulmonary disease and control identifies potential novel host factors post-influenza A virus infection

“…The primary human bronchial epithelial cells were expanded and cultured as described previously ( Berthold et al, 2022 ; Mastalerz et al, 2022 ). For more details, refer to the online Supplementary material .…”

“…For phBECs, immunofluorescence quantification was performed using the Imaris 7.4.0 software (Bitplane). Briefly, z-stack images of stained transwell membranes were generated by fluorescent microscopy, and individual cells were analyzed for the positivity of specific markers, largely as described previously ( Schamberger et al, 2015 ; Mastalerz et al, 2022 ). For patient lung sections, quantification was performed using NIS Elements version 5.41.00 (Nikon Solutions Co., Ltd.) as follows: the apical area of the bronchus was selected above the bronchial epithelial nuclear layer and along the apical surface to yield the bronchial region of interest (ROI).…”

“…Primary human bronchial epithelial cells (phBECs) differentiated at the air–liquid interface (ALI) represent a powerful tool to assess the airway-specific first-line defense against external stimuli. The resulting organotypic bronchoepithelium closely resembles the in vivo airway and is frequently used in studies of toxicology, infection, and drug delivery ( Pezzulo et al, 2011 ; Lenz et al, 2014 ; Singanayagam et al, 2019 ; Berthold et al, 2022 ; Mastalerz et al, 2022 ). The fully differentiated epithelium, obtained 4 weeks after the airlift, contains all major bronchial epithelial cell types and displays physiologically important functional properties like mucus secretion and ciliary beating ( Lodes et al, 2020 ).…”

Effects of immunophilin inhibitors and non-immunosuppressive analogs on coronavirus replication in human infection models

“…All phBECs were from patients undergoing lung tumor resections and were isolated from histologically normal regions adjacent to the resected lung tumors. Isolation and expansion of phBECs were performed as described previously ( Mastalerz et al., 2022 ). The study was approved by the local ethics committee of the Ludwig-Maximilians University of Munich, Germany (Ethic vote #19-630).…”

“…phBECs from three different donors were cultured as described previously ( Schamberger et al., 2015 ; Mastalerz et al., 2022 ). Cells were first expanded in p2 in PneumaCult™-ExPlus medium (Stemcell Technologies, Vancouver, Canada) supplemented with 1× Supplement, 1% penicillin/streptomycin, and 0.1% hydrocortisone in a 100 mm dish until reaching 85% confluency.…”