Continuous mucociliary transport by primary human airway epithelial cells in vitro

Abstract

Mucociliary clearance (MCC) is an important innate defense mechanism that continuously removes inhaled pathogens and particulates from the airways. Normal MCC is essential for maintaining a healthy respiratory system, and impaired MCC is a feature of many airway diseases, including both genetic (cystic fibrosis, primary ciliary dyskinesia) and acquired (chronic obstructive pulmonary disease, bronchiectasis) disorders. Research into the fundamental processes controlling MCC, therefore, has direct clinical application, but has been limited in part due to the difficulty of studying this complex multicomponent system in vitro. In this study, we have characterized a novel method that allows human airway epithelial cells to differentiate into a mucociliary epithelium that transports mucus in a continuous circular track. The mucociliary transport device allows the measurement and manipulation of all features of mucociliary transport in a controlled in vitro system. In this initial study, the effect of ciliary beat frequency and mucus concentration on the speed of mucociliary transport was investigated.

Keywords: airway; cilia; human; mucociliary; mucus.

Fig. 1.

A, left: illustration of the mucociliary transport (MCT) device (MCTD) from above. The MCTD track has an outer diameter of ∼2.3 cm and an inner diameter of ∼1.5 cm, with a track width of ∼4 mm. Right: photograph of an MCTD showing the central cylinder and the outer track. Bottom: illustration of the MCTD in cross section. The human airway epithelial cells (blue) are cultured on a porous membrane at the air/liquid interface above the culture media (red). Drawings are not to scale. B: paraffin sections of a well-differentiated MCTD stained with hematoxylin and eosin (left) or alcian blue-periodic acid Schiff (right) showing the presence of ciliated and secretory cells. Scale bar = 10 μm. C: composite image showing the motion of 3-μm fluorescent particles in the MCTD. Images were taken over a period of 7 s.

Fig. 2.

Orientation of ciliary beating in the MCTD. A: the overall direction of ciliary beating from 6 individual MCTD cultures (shaded lines) was plotted in relation to the direction of MCT. MCT is to the right (x-axis). The overall direction of ciliary activity (solid line) was closely aligned with the direction of MCT, with a vector magnitude of 0.69 and circular standard deviation of 0.86. B–G: orientation of ciliary beating in the 6 individual cultures included in A. Shaded lines indicate orientation of individual ciliated cells from 8 videos per culture, whereas solid lines indicate the overall orientation.

Fig. 3.

MCT speed as a function of ciliary beat frequency (CBF). CBF was varied by changing the temperature of the MCTD in 3 separate protocols. A: cultures were allowed to cool from 37 to 25°C. B: cultures were cooled on ice and then heated to 37°C. C: cultures were incubated at defined temperatures from 24.5 to 39.5°C in a stepwise fashion. In each experiment, the speed of MCT increased with increasing CBF in a linear fashion. Figure shows MCT plotted against CBF from one representative experiment. Each point represents the average of 9–10 measurements; error bars indicate SD. D, E, and F: the corresponding raw data, respectively. In each protocol, MCT varied directly in response to changes in CBF.

Fig. 4.

Relationship between mucin concentration and MCT speed. Three individual human bronchial epithelial (HBE) cultures (diamonds, squares, triangles) demonstrating continuous circular transport were washed, and different concentrations of purified bovine submaxillary mucin (BSM) were added to the apical surface. After a brief equilibration period, MCT was measured. Each culture was studied with four different concentrations of BSM; each point represents transport measured from 4 separate videos. The data demonstrate a linear dependence of MCT on mucin concentration (solid black line; r² = 0.86). Inset: cilary beat frequency was determined from the same cultures. Each shaded symbol represents whole field analysis of a single video, the solid circles represent the average of the 3 cultures, and the thick solid line indicates the least squares regression curve. Values are means ± SE.

Fig. 5.

Effect of forskolin stimulation on CBF and MCT. Baseline measures of CBF and MCT speed were obtained, and cultures were treated basolaterally with control solution or 10 μM forskolin, and the changes in CBF and MCT speed were measured (open bars, control; solid bars, forskolin). Each culture (n = 4; 3–4 videos per culture) was studied under both conditions. Values are means ± SE. #P < 0.0006 and *P < 0.002, compared with control.