A novel method for live imaging of human airway cilia using wheat germ agglutinin

Abstract

Multiciliated epithelial cells in the airway are essential for mucociliary clearance. their function relies on coordinated, metachronal and directional ciliary beating, appropriate mucus secretion and airway surface hydration. However, current conventional methods for observing human airway ciliary movement require ciliated cells to be detached from airway tissues. Determining the directionality of cilia is difficult. We developed a novel method to stain airway epithelial cilia to observe their movement without releasing ciliated cells. Human tracheae were obtained from patients (n = 13) who underwent laryngectomies to treat malignancies or swallowing disorders. The tracheae were treated with fluorescently labeled wheat germ agglutinin, which interacts with the acidic mucopolysaccharides present on the cilia. epithelial surfaces were observed using an epifluorescence microscope equipped with a water-immersion objective lens and a high-speed camera. Ciliary movement was observable at 125 fps (13/13 samples). Ciliated cells in close proximity mostly exhibited well-coordinated ciliary beats with similar directionalities. These findings indicated that wheat germ agglutinin renders ciliary beats visible, which is valuable for observing human airway ciliary movements in situ.

Lectin from Triticum vulgaris (WGA) Inhibits Infection with SARS-CoV-2 and Its Variants of Concern Alpha and Beta

“…WGA binds specifically to N-Acetyl-D-glucosamine (GlcNAc) and was shown to interact with sialic acid residues [ 53 ]. Due to its binding profile, WGA is widely used to label cell membranes and tissues in scientific imaging [ 54 , 55 ], and was shown to detect specific Gram-positive and Gram-negative bacteria [ 56 ]. It expresses antifungal activity [ 57 ] and interacts with immune cells in several ways, such as inhibiting T lymphocyte proliferation [ 58 , 59 ].…”

Structure-function relationships of mucociliary clearance in the human airways

“…We measured live ciliary beat and particle clearance 10,21 in freshly isolated airway epithelial tissue from respiratory tree branching generation (BG) 0 through BG6 from 3 human whole lungs with no prior history of chronic lung disease, and BG0 through BG5 from healthy rat whole lungs ( Fig. 1a ; for detailed donor information and numbers used in subsequent results see Supplementary Tables S1 & S2).…”

“…In some samples, ciliary beat was recorded with phase contrast at 100-200 frames per second (fps) using an inverted Leica microscope equipped with a 40x (NA 0.8) objective and a PCO Edge 4.2 high speed camera (Excelitas Technologies) operated with the micromanager plugin (ImageJ). Cilia were also live-stained with fluorescent-dye conjugated wheat germ agglutinin (ThermoFisher; 20 minute incubation in dilution of 1:200) 15 , and 1-µm fluorescent tracer particles were added to the bath, such that live ciliary beat and particle clearance could be recorded in the same field of view for 10 seconds (ciliary beat: 15-33 fps; particle trajectories 8-33 fps) using epifluorescence imaging as previously described 48 . Two to four FOVs with visible CBF were recorded from each sample.…”

“…However, our understanding of impaired MCC in disease remains limited by our incomplete knowledge of the mechanical structure-function relationships that link secretory and ciliated cell organization to MCC in humans 3 since most in vivo studies rely on animal models [12][13][14] . Data on live ciliary beat and clearance function are challenging to obtain in humans and remain rare in literature 15 . Secretory and ciliated cell organization at the epithelial surface remains equally understudied since conventional histology best reveals cross-sectional, instead of luminal, tissue architecture.…”

Hydrodynamic instability and flow reduction in polymer brush coated channels

“…From the synthetic point of view they can be manipulated under many respects (e.g. polymer species, surface geometry, grafting architecture) in order to finely tune the physico-chemical properties of a desired surface (the grafted substrate) [1][2][3][4]; however, they also constitute the puzzling ingredient of many mammalian organs [5][6][7]. In the first case, experimentalists exploit the brush equilibrium features and the versatility of the polymerization process to control its responsivenesses at different electro-chemical stimuli.…”