An Optimised Human Cell Culture Model for Alveolar Epithelial Transport

PLoS One. 2016 Oct 25;11(10):e0165225. doi: 10.1371/journal.pone.0165225. eCollection 2016.


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.

MeSH terms

  • A549 Cells
  • Cell Culture Techniques / methods*
  • Cell Line
  • Chloride Channels / metabolism
  • Colforsin / pharmacology
  • Culture Media / chemistry
  • Epithelial Cells / cytology*
  • Epithelial Cells / metabolism
  • Homeostasis
  • Humans
  • Ion Transport*
  • Models, Biological
  • Pulmonary Alveoli / cytology*
  • Pulmonary Alveoli / metabolism
  • Sodium Channels / metabolism


  • Chloride Channels
  • Culture Media
  • Sodium Channels
  • Colforsin

Grant support

This research was supported by: 1) the Marsden Fund (grant UOA1411) by the Royal Society of New Zealand (Recipient: VS),; 2) the Maurice Wilkins Centre for Molecular Biodiscovery by the Maurice Wilkins Centre (Recipient: VS),; 3) the University of Auckland’s Faculty Research Development Fund and Cross Faculty Research Initiatives by the University of Auckland (Recipient: VS),; 4) University of Auckland Doctoral Scholarship (Recipient: HR), The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.