A comparative study ofin vitroair-liquid interface culture models of the human airway epithelium evaluating cellular heterogeneity and gene expression at single cell resolution

Abstract

The airway epithelium is composed of diverse cell types with specialized functions that mediate homeostasis and protect against respiratory pathogens. Human airway epithelial cultures at air-liquid interface (HAE) are a physiologically relevant in vitro model of this heterogeneous tissue, enabling numerous studies of airway disease. HAE cultures are classically derived from primary epithelial cells, the relatively limited passage capacity of which can limit experimental methods and study designs. BCi-NS1.1, a previously described and widely used basal cell line engineered to express hTERT, exhibits extended passage lifespan while retaining capacity for differentiation to HAE. However, gene expression and innate immune function in HAE derived from BCi-NS1.1 versus primary cells have not been fully characterized. Here, combining single cell RNA-Seq (scRNA-Seq), immunohistochemistry, and functional experimentation, we confirm at high resolution that BCi-NS1.1 and primary HAE cultures are largely similar in morphology, cell type composition, and overall transcriptional patterns. While we observed cell-type specific expression differences of several interferon stimulated genes in BCi-NS1.1 HAE cultures, we did not observe significant differences in susceptibility to infection with influenza A virus and Staphylococcus aureus. Taken together, our results further support BCi-NS1.1-derived HAE cultures as a valuable tool for the study of airway infectious disease.

In-silicodocking platform with serine protease inhibitor (SERPIN) structures identifies host cysteine protease targets with significance for SARS-CoV-2

“…Given the importance of airway epithelial cells in producing SERPINs during SARS-CoV-2 infection ( Figure 1 ), we next used polarized human airway epithelial cultures (HAEC) to study the expression patterns of SERPINs in a controlled environment in vitro . The HAEC model closely mimics the human bronchial epithelium, encompassing various cell types (basal, ciliated, and secretory), architectural features, and a secreted extracellular environment that includes mucus and proteases ( Figure 2a, b ) 33 . We infected HAEC with a panel of clinically relevant respiratory viruses ( Figure 2b ), all of which feature proteolytic steps in their life cycles.…”

In vitro modelling of bacterial pneumonia: a comparative analysis of widely applied complex cell culture models

“…Another less common bronchial cell line is BCi-NS1.1, a basal cell immortalized-nonsmoker 1 cell line, used by Prescott et al. ( 2023 ) to investigate S. aureus infection in ALI cultured models (Prescott et al 2023 ). These cells retain many original characteristics of primary cells and give rise to cells with goblet and club cell character, as well as mucus-producing and ciliated cells (Walters et al 2013 , Prescott et al 2023 ).…”

“…( 2023 ) to investigate S. aureus infection in ALI cultured models (Prescott et al 2023 ). These cells retain many original characteristics of primary cells and give rise to cells with goblet and club cell character, as well as mucus-producing and ciliated cells (Walters et al 2013 , Prescott et al 2023 ). Remarkable is also the use of Caco-2 cells by Nair et al ( 2016 ), as these cells originate from human colorectal adenocarcinoma, and are typically used as a ALI model of the intestinal barrier (Nair et al 2016 ).…”

“…CFU plating of supernatant can be useful in ALI or LOC models to track bacterial migration from one compartment to another, but should not be used to estimate a total bacterial count, as bacterial adhesion and internalization will lead to underestimation of bacterial presence (Iakobachvili et al 2022 , Tang et al 2022 , Ma et al 2023 ). Most bacterial quantification techniques, however, require an endpoint step such as cell lysis (Kotze et al 2021 , Thorn et al 2021 , Prescott et al 2023 ). Release of bacterial material, including RNA and exotoxins, could be studied by analysing cell supernatant by similar techniques as described above.…”

“…Several studies also reported a general viable count without distinguishing between internal or external, adherent, or nonadherent bacteria (McMichael et al 2005 , Garnett et al 2013 , Prescott et al. 2023 ).…”