title: 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.
authors: Auckland Bioengineering Institute, University of Auckland, Auckland, New Zealand.|School of Biological Sciences, University of Auckland, Auckland, New Zealand.
authors: Hui
authors: http://orcid.org/0000-0002-0166-8614
authors: H
authors: Ren
authors: School of Biological Sciences, University of Auckland, Auckland, New Zealand.|Centre for Brain Research, University of Auckland, Auckland, New Zealand.|Brain Research New Zealand, Rangahau Roro Aotearoa, New Zealand.
authors: Nigel P
authors: NP
authors: Birch
authors: Auckland Bioengineering Institute, University of Auckland, Auckland, New Zealand.|Department of Engineering Science, University of Auckland, Auckland, New Zealand.|Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand.
authors: Vinod
authors: V
authors: Suresh
mesh_terms: D000072283:A549 Cells; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D003470:Culture Media / Q000737:chemistry; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D017136:Ion Transport; D008954:Models, Biological; D011650:Pulmonary Alveoli / Q000166:cytology* / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism
chemical_list: D018118:Chloride Channels; D003470:Culture Media; D015222:Sodium Channels; D005576:Colforsin