The Cell Line NCl-H441 Is a Useful in Vitro Model for Transport Studies of Human Distal Lung Epithelial Barrier

Abstract

The lack of a well characterized, continuously growing in vitro model of human distal lung epithelial phenotype constitutes a serious limitation in the area of inhalation biopharmaceutics, particularly in the context of transepithelial transport studies. Here, we investigated if a human lung adenocarcinoma cell line, NCl-H441, has potential to serve as an in vitro model of human distal lung epithelium. The development of barrier properties was studied by immunocytochemistry (ICC) against the junction proteins zonula occludens protein 1 (ZO-1) and E-cadherin and measurement of transepithelial electrical resistance (TEER). Moreover, transport studies with the paracellular marker compounds fluorescein sodium and fluorescein isothiocyanate (FITC)-labeled dextrans of molecular weights ranging from 4 to 70 kDa were carried out. The expression of P-glycoprotein (P-gp; ABCB1) and organic cation transporters (OCT/Ns; SLC22A1-A5) was investigated by ICC and immunoblot. P-gp function was assessed by monolayer release and bidirectional transport studies using rhodamine 123 (Rh123) and the inhibitors verapamil and LY335979. Uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) was measured, in order to assess organic cation transporter function in vitro. Furthermore, the inhibitory potential of several organic cations on ASP(+) uptake was studied. NCl-H441 cells, when grown under liquid-covered conditions, formed confluent, electrically tight monolayers with peak TEER values of approximately 1000 Ω·cm(2), after 8-12 days in culture. These monolayers were able to differentiate paracellularly transported substrates according to their molecular weight. Presence of P-gp, OCT1, OCT2, OCT3, OCTN1, and OCTN2 was confirmed by Western blot and ICC and was similar to data from freshly isolated human alveolar epithelial cells in primary culture. Rh123 release from NCI-H441 monolayers was time-dependent and showed low, albeit significant attenuation by both inhibitors. In transport studies, Rh123 exhibited net secretion, which again was inhibitable by bona fide P-gp modulators. The uptake of ASP(+) was time- and temperature-dependent with Km = 881.2 ± 195.3 μM and Vmax = 2.07 ± 0.26 nmol/min/mg protein. TEA, amantadine, quinidine, and verapamil significantly inhibited ASP(+) uptake into NCl-H441 cells, whereas the effect of d- and l-carnitine and ergothioneine, two OCTN substrates, was less pronounced. NCl-H441 cells are the first cell line of human distal lung epithelial origin with the ability to form monolayers with appreciable barrier properties. Moreover, drug transporter expression and activity in NCl-H441 cells was consistent with what has been reported for human alveolar epithelial cells in primary culture.

Organic cation transporter function in different in vitro models of human lung epithelium

“…OCT expression pattern determined by Western blot Salomon et al, 2014) and the now studied TEA uptake rate were quite similar in ATI-like and A549 cells. Nevertheless, were the highest number of significantly different pharmacological effects on TEA uptake observed when comparing the two cell types.…”

“…Considering that expression patterns of OCT between the two cell types were very close when assessed with molecular biological techniques (Salomon et al, 2014), NCl-H441 epithelial cells seem to exhibit profound similarities to primary cells in their spatial expression and organic cation transporter function.…”

“…type II cell) and bronchiolar (i.e. club cell) epithelial phenotype (Hermanns et al, 2005;Salomon et al, 2014).…”

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

“…These cells, which were derived from a papillary adenocarcinoma of the lung, have features characteristic of Club cells (Gazdar et al, 1990) and have been used as a model of bronchiolar epithelium (Bruce et al, 2009). However, more recently, it has been found that culture of H441 cells on semi-permeable filter supports results in formation of electrically tight monolayers (peak TEER values of approximately 1000 Ω cm 2 after 8-12 days in culture) which are able to differentiate paracellularly transported substrates according to their molecular weight (Salomon et al, 2014). In this model, the cells express a number of key drug transporters and P-glycoprotein function was found to be comparable to human primary ATI-like pneumocytes.…”

Influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions

“…NCl-H441 cells (which morphologically display characteristics of type II pneumocytes and club cells) [25,26] were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI (Gibco, Grand Island, NY, USA) with 10% fetal calf serum (FCS) (Sigma, St Louis, MO, USA) and 1% penicillin-streptomycin (Lonza, Basel, Switzerland). ISO-HAS-1 cells (which have previously been described to display the key features of endothelial cells) [27] were cultured in the same conditions.…”