Immortalisation of primary human alveolar epithelial lung cells using a non-viral vector to study respiratory bioreactivity in vitro

Abstract

To overcome the scarcity of primary human alveolar epithelial cells for lung research, and the limitations of current cell lines to recapitulate the phenotype, functional and molecular characteristics of the healthy human alveolar epithelium, we have developed a new method to immortalise primary human alveolar epithelial lung cells using a non-viral vector to transfect the telomerase catalytic subunit (hTERT) and the simian virus 40 large-tumour antigen (SV40). Twelve strains of immortalised cells (ICs) were generated and characterised using molecular, immunochemical and morphological techniques. Cell proliferation and sensitivity to polystyrene nanoparticles (PS) were evaluated. ICs expressed caveolin-1, podoplanin and receptor for advanced glycation end-products (RAGE), and most cells were negative for alkaline phosphatase staining, indicating characteristics of AT1-like cells. However, most strains also contained some cells that expressed pro-surfactant protein C, classically described to be expressed only by AT2 cells. Thus, the ICs mimic the cellular heterogeneity in the human alveolar epithelium. These ICs can be passaged, replicate rapidly and remain confluent beyond 15 days. ICs showed differential sensitivity to positive and negatively charged PS nanoparticles, illustrating their potential value as an in vitro model to study respiratory bioreactivity. These novel ICs offer a unique resource to study human alveolar epithelial biology.

Figure 1

TEM images of LF particles alone (a,b) and in nanomeric complex with DNA (c,d).

Figure 2

Relative transcript levels of RAGE (a), caveolin-1 (b) and SP-C (c) in ICs and TT1 cell line. Values are given as means ± SD. Stars indicate significant differences (p < 0.05) among groups according to the Kruskal–Wallis followed by the Dunn’s post hoc test. n = 6 replicates per sample.

Figure 3

Immunoblotting analysis of RAGE, caveolin-1, podoplanin and SP-C in ICs, TT1 cell line and primary AT2 cells. Loading protein concentrations were adjusted to 50 μg per sample.

Figure 4

Immunofluorescent labelling of podoplanin (green) in AT2 cells (a), TT1 cell line (b) and ICs (c,d). Podoplanin is present in most of the cells but not in all of them (white arrows). Cell nuclei are stained blue. Bars = 50 µm.

Figure 5

Alkaline phosphatase staining showing AP-positive AT2 cells (a) and negative TT1 cells (b). Some ICs were AP-negative (c) whereas others were mostly AP-negative showing scattered AP-positive cells (arrows) (d). Bars = 50 µm.

Figure 6

TEM images of ICs showing a flattened thin morphology (a), membrane invaginations (caveolae, arrows) (b), clathrin-coated vesicles (arrows) (c) and numerous endosomal vesicles (arrows) (d). Some cells showed a different morphology with the presence of apical microvilli (arrows) (e), cytoplasmic inclusions resembling lamellar bodies (f) and tight junctions (g).

Figure 7

Cell proliferation given as cell index of ICs and TT1 cells after 1 (a), 5 (b), 10 (c) and 15 (d) days (media changed every 3 days).

Figure 8

Cell viability (MTT assay) of TT1 cells (a) and ICs (b–d) exposed to 50 nm PS-NH₂ and PS-COOH particles for 24 h. Data are given as percentage with respect to controls (means ± SD). Stars indicate significant differences (p < 0.05) in treated cells with respect to controls according to the Kruskal–Wallis followed by the Dunn’s post hoc test. n = 6 replicates per treatment. LC50 values were calculated through Probit analysis.