An Optimised Human Cell Culture Model for Alveolar Epithelial Transport

Abstract

Robust and reproducible in vitro models are required for investigating the pathways involved in fluid homeostasis in the human alveolar epithelium. We performed functional and phenotypic characterisation of ion transport in the human pulmonary epithelial cell lines NCI-H441 and A549 to determine their similarity to primary human alveolar type II cells. NCI-H441 cells exhibited high expression of junctional proteins ZO-1, and E-cadherin, seal-forming claudin-3, -4, -5 and Na+-K+-ATPase while A549 cells exhibited high expression of pore-forming claudin-2. Consistent with this phenotype NCI-H441, but not A549, cells formed a functional barrier with active ion transport characterised by higher electrical resistance (529 ± 178 Ω cm2 vs 28 ± 4 Ω cm2), lower paracellular permeability ((176 ± 42) ×10−8 cm/s vs (738 ± 190) ×10−8 cm/s) and higher transepithelial potential difference (11.9 ± 4 mV vs 0 mV). Phenotypic and functional properties of NCI-H441 cells were tuned by varying cell seeding density and supplement concentrations. The cells formed a polarised monolayer typical of in vivo epithelium at seeding densities of 100,000 cells per 12-well insert while higher densities resulted in multiple cell layers. Dexamethasone and insulin-transferrin-selenium supplements were required for the development of high levels of electrical resistance, potential difference and expression of claudin-3 and Na+-K+-ATPase. Treatment of NCI-H441 cells with inhibitors and agonists of sodium and chloride channels indicated sodium absorption through ENaC under baseline and forskolin-stimulated conditions. Chloride transport was not sensitive to inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) under either condition. Channels inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) contributed to chloride secretion following forskolin stimulation, but not at baseline. These data precisely define experimental conditions for the application of NCI-H441 cells as a model for investigating ion and water transport in the human alveolar epithelium and also identify the pathways of sodium and chloride transport.

Pro-inflammatory effects of crystalline- and nano-sized non-crystalline silica particles in a 3D alveolar model

“…However, to more properly address the importance of IL-1 in 3D co-culture systems, there is a need to optimize the models to have tight alveolar-endothelial cell barriers. To achieve a more functional lung cell barrier other epithelial lung cell lines need to be introduced to replace A549 cells, to better mimic the in vivo situation [73,[76][77][78][79]. As A549 cells also are known to be less responsive to different toxicants [80] than many other lung epithelial cell lines, and in particular compared to primary type 2 cells, replacement of A549 cells with other lung epithelial cell types, might enhance the sensitivity of the model system.…”

Zinc Chelation Specifically Inhibits Early Stages of Dengue Virus Replication by Activation of NF-κB and Induction of Antiviral Response in Epithelial Cells

“…We first examined the barrier properties of two epithelial cell lines Caco-2 (colon) and A549 (lung) by growing these cells on transwell inserts for 4 days and measuring the trans-epithelial electrical resistance (TEER) every day. Caco-2 cells have been reported to have higher expression of tight junction proteins and undergo differentiation whereas A549 cells have lower TEER values and do not undergo differentiation (27)(28)(29)(30). As expected, the basal levels of TEER was about 200-fold lower in A549 cells as compared to Caco-2 on day 2, however, both cell lines showed an increase in the TEER values with time in culture and started to plateau by day 4 (Supplementary Figures 1A,B).…”

Modulation of cell adhesion and migration by poly-dispersed-acid-functionalized-single-walled carbon nanotubes in lung epithelial cells

“…These results confirmed the Western blot results for these two markers. Since highly hydrophobicCNTsexist as huge insoluble agglomerates in aqueous media, a poly-dispersed acid-functionalized form of single-walled carbon nanotubes (AF-SWCNTs) was used in our study and their effects examined on two lung epithelial cell lines [a mouse (LA4) and a human (A549)], as both these cell lines have extensively been used as models for studying the lung epithelial cells function (Sonar et al 2010,Ren et al 2016.While both cell lines efficiently internalized AF-SWCNTs and showed 100% positivity for AF-SWCNTs after 12 hours of incubation, A549 accumulated more AF-SWCNTs as indicated by significantly higher MFI values (Figure 1). Confocal laser microscopy and the Z-sectioning studies reveal that, most of the internalized carbon nanotubes were localized in the cytoplasmic space while a much smaller number transgressed nuclear membrane and was visible inside the nucleus.…”