Evaluation of differentiated human bronchial epithelial cell culture systems for asthma research

Abstract

The aim of the current study was to evaluate primary (human bronchial epithelial cells, HBEC) and non-primary (Calu-3, BEAS-2B, BEAS-2B R1) bronchial epithelial cell culture systems as air-liquid interface- (ALI-) differentiated models for asthma research. Ability to differentiate into goblet (MUC5AC+) and ciliated (β-Tubulin IV+) cells was evaluated by confocal imaging and qPCR. Expression of tight junction/adhesion proteins (ZO-1, E-Cadherin) and development of transepithelial electrical resistance (TEER) were assessed. Primary cells showed localised MUC5AC, β-Tubulin IV, ZO-1, and E-Cadherin and developed TEER with, however, a large degree of inter- and intradonor variation. Calu-3 cells developed a more reproducible TEER and a phenotype similar to primary cells although with diffuse β-Tubulin IV staining. BEAS-2B cells did not differentiate or develop tight junctions. These data highlight the challenges in working with primary cell models and the need for careful characterisation and selection of systems to answer specific research questions.

Figure 1

Development of transepithelial electrical resistance (TEER) in cells grown at (ALI). Different primary cells and cell lines were cultured at ALI over 21–28 days. TEER was measured every 2-3 days. Results from three separate experiments are shown for each cell line/donor, six replicates per experiment. HBEC D1 is Donor 1 and HBEC D2 is Donor 2. Error bars show standard deviation.

Figure 2

Phase contrast images of cells at ALI. Different primary cells and cell lines were cultured at ALI over 21–28 days. Phase contrast images were taken at 21 days. Representative images are from three independent experiments.

Figure 3

Immunofluorescent confocal imaging of differentiation markers. Localisation patterns of β-Tubulin IV, MUC5AC, and the Mouse IgG Isotype control at 28 days ALI were evaluated as described in the methods section. Single Z-slices are shown representing maximum intensity observed, with the corresponding Z-stack image below for β-Tubulin IV and MUC5AC. Scale bar represents 50 μm. Representative images are from three independent experiments. HBEC images were taken from experiments with low TEER (Figure 1).

Figure 4

Immunofluorescent confocal imaging of tight junction proteins. Localisation patterns of ZO-1, E-Cadherin, and the Rabbit IgG Isotype control at 28 days ALI were evaluated as described in the methods section. Images shown are single Z-stack slices representing maximum intensity observed with the corresponding Z-stack image below and are of matched fields using dual staining. The corresponding Mouse Isotype control for E-Cadherin can be seen in Figure 3. Scale bar represents 50 μm. Representative images are from three independent experiments. Images for HBEC Donor 1 were taken from an experiment reaching high TEER (>350 Ω·cm²), whilst Donor 2 images were taken from an experiment reaching low TEER (Figure 1).

Figure 5

mRNA expression of differentiation and tight junction markers. Primary cells and cell lines were cultured at ALI over 21–28 days. RNA was extracted at days 7, 14, and 21 during ALI differentiation for each cell line or donor. Expression of MUC5AC (a), β-Tubulin IV (b), E-Cadherin (c), and ZO-1 (d) was measured. Data are normalised to the housekeeping gene HPRT1. Data are representative of two independent experiments. Error bars show standard deviation. Yellow, red, and blue bars represent expression at days 7, 14, and 21 post-ALI, respectively.