Temporal differentiation of bovine airway epithelial cells grown at an air-liquid interface

Abstract

There is an urgent need to develop improved, physiologically-relevant in vitro models of airway epithelia with which to better understand the pathological processes associated with infection, allergies and toxicological insults of the respiratory tract of both humans and domesticated animals. In the present study, we have characterised the proliferation and differentiation of primary bovine bronchial epithelial cells (BBECs) grown at an air-liquid interface (ALI) at three-day intervals over a period of 42 days from the introduction of the ALI. The differentiated BBEC model was highly representative of the ex vivo epithelium from which the epithelial cells were derived; a columnar, pseudostratified epithelium that was highly reflective of native airway epithelium was formed which comprised ciliated, goblet and basal cells. The hallmark defences of the respiratory tract, namely barrier function and mucociliary clearance, were present, thus demonstrating that the model is an excellent mimic of bovine respiratory epithelium. The epithelium was fully differentiated by day 21 post-ALI and, crucially, remained healthy and stable for a further 21 days. Thus, the differentiated BBEC model has a three-week window which will allow wide-ranging and long-term experiments to be performed in the fields of infection, toxicology or general airway physiology.

Figure 1

Histological assessment of BBEC differentiation over time. Bronchial epithelial cell cultures were grown for the indicated number of days at an ALI (relative to the establishment of the ALI), fixed and paraffin embedded using standard histological techniques. Samples of ex vivo bronchial epithelial tissue were similarly fixed and embedded. Sections were cut, deparaffinised and stained using (A) H&E and (B) immunohistochemistry with an anti-p63 antibody labelling the nuclei of basal cells (brown staining). Representative images are shown of ex vivo bronchial epithelium and BBECs grown for 3, 12, 21 and 42 days post-ALI (see also Figs S1 and S2). Quantitative analysis (using ImageJ) was performed of histological sections of BBEC layers fixed at three-day intervals ranging from day −3 to day 42 post-ALI (see Fig. S1); the epithelial thickness (C) and number of cell layers (D) comprising the epithelium were measured. For each insert, three measurements were taken (left, centre and right) in each of five randomised 400x fields of view evenly distributed across the strand; three inserts were analysed per time-point and the data represents the mean +/−standard deviation from tissue derived from three different animals (n = 9). Ordinary One-way ANOVA statistical analyses with post-test for linear trend demonstrated significant (P ≤ 0.05) increasing trends over time for both epithelial thickness (C) and the number of cell layers (D).

Figure 2

Comparison of cellular composition, morphology and polarisation of differentiated BBECs and ex vivo bronchial epithelium using fluorescent immunohistochemistry. Ex vivo bronchial epithelium dissected from the donor animal before cell extraction (i) and day 21 post-ALI BBEC cultures (ii) were fixed and paraffin-embedded using standard histological techniques. Sections were cut, deparaffinised and immunohistochemically stained to identify markers of specific epithelial cell-type as follows: cilia (ciliated cells) - red; Muc5AC (mucus and goblet cells) - green; p63 (basal cells) - blue; nuclei - grey.

Figure 3

Tight-junction formation and changes in barrier function in BBEC cultures over time. Bronchial epithelial cell cultures were grown for the indicated number of days at an ALI (relative to the establishment of the ALI) and fixed in situ on the membranes. In (A) tight-junction formation was subsequently assessed using immunofluorescence staining of the tight-junction protein ZO-1 (tight junctions - green; nuclei - blue). Representative images are shown of BBECs at −3, 12, 21 and 42 days post-ALI (see also Fig. S4). In (B) junctional complexes of ex vivo bronchial epithelial tissue and day 21 post-ALI BBEC cultures were assessed by TEM. Tight-junctions (T), adherens junctions (A) and desmosomes (D) were present along the apicolateral borders of epithelial cells in both the ex vivo tissue and BBEC cultures. In addition, membranous interdigitations (arrow) were observed between adjacent cells in the BBEC cultures. In (C) tight-junction integrity during the course of epithelial cell proliferation and differentiation was assessed by measuring the TEER of BBEC cultures until day 42 post-ALI. Nine inserts were analysed per time-point and the data represents the mean +/−standard deviation from tissue derived from three different animals (n = 27).

Figure 4

Cilia formation in BBEC cultures over time. Bronchial epithelial cell cultures were grown for the indicated number of days at an ALI (relative to the establishment of the ALI) before fixation. The BBEC cultures were subsequently processed to assess ciliation using (A) H&E staining of histological sections (arrowheads indicate ciliated cells), (B) immunofluorescence staining (cilia - green; F-actin - red; nuclei - blue) and (C) SEM. Representative images are shown of BBECs grown for 0, 12, 21 and 41 days post-ALI (see also Figs S1, S5 and S6). Quantitative analysis (using ImageJ) of ciliation of BBEC cultures fixed at three-day intervals ranging from day −3 to day 42 post-ALI was performed (D) by counting the number of ciliated cells per field of view in H&E-stained sections (see Fig. S1) and (E) using fluorescence intensity thresholding of immunostained cultures (see Fig. S5). In (D) for each insert, ciliated cells were counted in each of five randomised 400x fields of view evenly distributed across the strand. In (E) ciliation was quantified by measuring the area above a fluorescence intensity threshold in ImageJ; for each insert, five regions evenly distributed across the sample were measured. For all of the above quantifications, three inserts were analysed per time-point and the data represents the mean +/−standard deviation from tissue derived from three different animals (n = 9). Statistical significance was tested using an Ordinary one-way ANOVA: ns = not significant.

Figure 5

Mucus production and identification of goblet cells in BBEC cultures. Bronchial epithelial cell cultures were grown for 21 days at an ALI before fixation. The BBEC cultures were subsequently processed to assess mucus production and identify goblet cells using (A) immunofluorescence microscopy and (B) SEM. In (A) standard immunofluorescence imaging shows mucus and its association with goblet cells (i) (mucus [Muc5AC] - green; nuclei – blue); Z-stack orthogonal representation highlights surface-localisation of mucus (ii) (mucus [Muc5AC] - green; cilia - red; nuclei - blue). In (B) SEM revealed the presence of numerous globules of mucus and their close association with cilia ([i], arrowheads) and identified goblet cells in the act of extruding mucus ([ii], arrowhead).

Figure 6

Scanning electron microscopic assessment of ultrastructural changes in BBEC cultures over time. Bronchial epithelial cell cultures were grown until 42 days post-ALI and samples fixed at three day intervals and processed for SEM (see also Fig. S6). Representative micrographs show (i) the undifferentiated, flattened apical surface at day 0 post-ALI with extruding nuclei (arrowheads); (ii) microvilli (arrow) and microplicae (arrowhead) on undifferentiated cells at day 0 post-ALI; (iii) longer microvilli (arrowheads) on day 3 post ALI cells undergoing differentiation; (iv) early stages of cilia formation (arrowheads) on day 6 post-ALI cells undergoing differentiation; (v) numerous well-formed cilia on day 12 post ALI cells; (vi) maximum ciliation on day 24 post-ALI cells; (vii) goblet cells (arrowheads) present in day 21 post-ALI cultures; (viii) section of day 18 post-ALI epithelium showing pseudostratified structure; and (ix) microvilli (arrowheads) at base of cilia associated with day 36 post-ALI cells.

Figure 7

Ultra-structural comparison of cilia formation in ex vivo bronchial epithelium and differentiated BBECs using TEM. Ex vivo bronchial epithelium and day 21 post-ALI BBEC cultures were fixed and resin embedded, and ultrathin sections were cut and contrast stained. Comparisons are made of (A) longitudinal sections of cilia and basal bodies (arrowheads) and (B) transverse sections of cilia showing 9 + 2 axoneme arrangement (arrowheads).

Figure 8

Ultra-structural comparison of goblet cells and paracellular spaces in ex vivo bronchial epithelium and differentiated BBECs using TEM. Ex vivo bronchial epithelium and day 21 post-ALI BBEC cultures were fixed and resin embedded, and ultrathin sections were cut and contrast stained. Images highlight (A) goblet cells (arrows) containing numerous secretory vesicles (arrowheads) and (B) paracellular spaces (arrows) containing membranous interdigitations (arrowheads).