A Revised Protocol for Culture of Airway Epithelial Cells as a Diagnostic Tool for Primary Ciliary Dyskinesia

Abstract

Air–liquid interface (ALI) culture of nasal epithelial cells is a valuable tool in the diagnosis and research of primary ciliary dyskinesia (PCD). Ex vivo samples often display secondary dyskinesia from cell damage during sampling, infection or inflammation confounding PCD diagnostic results. ALI culture enables regeneration of healthy cilia facilitating differentiation of primary from secondary ciliary dyskinesia. We describe a revised ALI culture method adopted from April 2018 across three collaborating PCD diagnostic sites, including current University Hospital Southampton COVID-19 risk mitigation measures, and present results. Two hundred and forty nasal epithelial cell samples were seeded for ALI culture and 199 (82.9%) were ciliated. Fifty-four of 83 (63.9%) ex vivo samples which were originally equivocal or insufficient provided diagnostic information following in vitro culture. Surplus basal epithelial cells from 181 nasal brushing samples were frozen in liquid nitrogen; 39 samples were ALI-cultured after cryostorage and all ciliated. The ciliary beat patterns of ex vivo samples (by high-speed video microscopy) were recapitulated, scanning electron microscopy demonstrated excellent ciliation, and cilia could be immuno-fluorescently labelled (anti-alpha-tubulin and anti-RSPH4a) in representative cases that were ALI-cultured after cryostorage. In summary, our ALI culture protocol provides high ciliation rates across three centres, minimising patient recall for repeat brushing biopsies and improving diagnostic certainty. Cryostorage of surplus diagnostic samples was successful, facilitating PCD research.

Properties of Non-Aminoglycoside Compounds Used to Stimulate Translational Readthrough of PTC Mutations in Primary Ciliary Dyskinesia

“…Time-lapse high-speed video microscopy (HSVM) at 37 °C was used to observe and measure the effect of NAGs on the ciliary beat frequency (CBF) as a surrogate for cilia health (with an in-house normal range of 11–20 Hz [ 36 , 37 ]). PNECs were seeded into 24-well culture plates, and using a motorized stage driven with time-lapse software, repeated hourly cilia measurements (at three x, y, z points corresponding to ciliated clusters of PNECs) were recorded for 24 h. Baseline CBFs were recorded prior to PNEC treatment with NAGs or controls.…”

“…Ciliated cells collected by nasal brushings from 3 healthy donors were seeded in a 24-well plate in Basal Epithelial Cell Growth medium (PromoCell, Heidelberg, Germany) [ 36 ]. After 24 h, the medium was replaced by Medium 199 (Sigma-Aldrich Corporation, Saint Louis, MO, USA) with different NAG concentrations, and cells were placed in the incubating chamber (37 °C) attached to the stage of the IX81 (Olympus, Shinjuku, Tokyo, Japan) motorized inverted microscope C-mounted to a high-speed video camera PC2 FASTCAM (Photron, Tokyo, Japan).…”

“…PNECs treated with 2 mg/mL of gentamicin demonstrated a 20% reduction in CBF (13.81 Hz ± 0.85 to 11.04 Hz ± 1.26), which sustained during the first 4 h after compound administration before returning to the baseline CBF (Figure 2, orange line). [36]. Please note that the Y axis starts from 50%.…”

“…The vectors used in the experiment contained one of six PTC mutations corresponding to those detected in Polish PCD patients (Table 1) or a corresponding wild-type DNA sequence (wt). [36]. Please note that the Y axis starts from 50%.…”

“…Per well, the localization of three regions of interest (ROIs) containing cell clusters with visible cilia were selected and x, y, z positions were programmed into an automated time-lapse system with a motorized stage (Xcellence rt software, version 2, build 4768, Olympus, Shinjuku, Tokyo, Japan). Ciliary movement at each ROI was recorded for 10 s at 250 frames per second at 1-h intervals for 24 h. To retrieve the ciliary beating frequency (CBF) of the cell clusters, recorded videos were analyzed by automated fast Fourier transform analysis (ImageJ software (https://imagej.net/, accessed on 21 February 2021) with a dedicated CBF Panel plug-in, courtesy of Peter Lackie from the University of Southampton, Southampton, UK) [36]. Individual data points were a mean of three ROIs and experiments were repeated independently with three different PNEC donor samples.…”