Methods for the assessment of human airway ciliary function

Jackson, Claire; Bottier, Mathieu

doi:10.1183/13993003.02300-2021

Cited by 7 publications

(9 citation statements)

References 72 publications

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“…Using high-speed video microscopy [34], ciliary beating was observed in the bronchioid model (movie E2). For bronchioids generated from 4 different patients, we determined the median ciliary beat frequency (CBF) across different fields of acquisition (figure 5a-b).…”

Section: Resultsmentioning

confidence: 99%

A novel in vitro tubular model to recapitulate features of distal airways: The bronchioid

Maurat,

Raasch,

Leipold

et al. 2023

Preprint

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BackgroundAirflow limitation is the hallmark of obstructive pulmonary diseases. While numerousin vitroairway models already exist, chronic obstructives diseases currently lack an 3D culture system enabling to understand how airway remodelling and the associated constrictive limitation develop. Here, the objective was to build a model of small airways, which would overcome the limitations of current culture systems.MethodsWorking with the Cellular Capsule Technology, we developed a so-called bronchioid model, using an innovative tubular cell-based assay and human bronchial adult stem cells derived from clinical samples.ResultsWe produced a tubular scaffold made of alginate gel, that drives the spontaneous self-organisation of lung epithelial cells. Fine control of the level of contraction is required to obtain a model of distal bronchiole, with physiologically relevant shape and size. 3D imaging and gene expression analysis of bronchioids made of primary bronchial epithelial cells demonstrates the tubular organization, the formation of epithelial junctions, as well as differentiation into ciliated and goblet cells. Ciliary beating is observed, at a decreased frequency in bronchioids made of cells from COPD patients. The bronchioid can be infected by rhinovirus. An air-liquid interface is introduced, that modulates gene expression.ConclusionWe provide here a proof of concept of a perfusable bronchioid, with proper mucociliary and contractile functions. Key advantages of our approach, such as the air-liquid interface, the lumen accessibility, the recapitulation of pathological features and possible assessment of clinically pertinent endpoints, will make our pulmonary organoid-like model a powerful tool for pre-clinical studies.

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Section: Resultsmentioning

confidence: 99%

A novel in vitro tubular model to recapitulate features of distal airways: The bronchioid

Maurat,

Raasch,

Leipold

et al. 2023

Preprint

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“…HSVA is a technique that uses light microscopy to observe respiratory cilia ex vivo and a high-speed video camera to record them [46]. Ciliary beat frequency (CBF), ciliary beat pattern (CBP), and effective mucociliary clearance are all measured in recorded videos [47].…”

Section: High-speed Video Microscopy Analysis (Hsva)mentioning

confidence: 99%

The RSPH4A Gene in Primary Ciliary Dyskinesia

Jesus-Rojas

Melendez-Montañez

Muñiz-Hernández

et al. 2023

IJMS

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The radial spoke head protein 4 homolog A (RSPH4A) gene is one of more than 50 genes that cause Primary ciliary dyskinesia (PCD), a rare genetic ciliopathy. Genetic mutations in the RSPH4A gene alter an important protein structure involved in ciliary pathogenesis. Radial spoke proteins, such as RSPH4A, have been conserved across multiple species. In humans, ciliary function deficiency caused by RSPH4A pathogenic variants results in a clinical phenotype characterized by recurrent oto-sino-pulmonary infections. More than 30 pathogenic RSPH4A genetic variants have been associated with PCD. In Puerto Rican Hispanics, a founder mutation (RSPH4A (c.921+3_921+6delAAGT (intronic)) has been described. The spectrum of the RSPH4A PCD phenotype does not include laterality defects, which results in a challenging diagnosis. PCD diagnostic tools can combine transmission electron microscopy (TEM), nasal nitric oxide (nNO), High-Speed Video microscopy Analysis (HSVA), and immunofluorescence. The purpose of this review article is to provide a comprehensive overview of current knowledge about the RSPH4A gene in PCD, ranging from basic science to human clinical phenotype.

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“…Measuring cilia beat frequency (CBF) has been a topic of interest for over a century and has advanced from early methods using a stroboscope 5 and photomultipliers 6 to recent methods using digital cameras and high-speed video microscopy [7][8][9][10][11] . Highspeed video microscopy enables direct observation of cilia beating and extraction of CBF data but manual counting is time-consuming and prone to human error.…”

Section: Introductionmentioning

confidence: 99%

“…FFT is limited to data with length equal to a power of 2 as it breaks down the function into many smaller equations of size ܰ/2 to simplify the calculation 13 . The available software for calculating CBF shown in Table 1 has recently been reviewed 14 . The main differences between existing tools lies in their handling of noise, which is particularly important when analysing samples with limited cilia movement.…”

Section: Introductionmentioning

confidence: 99%

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ciliR: an R package for determining ciliary beat frequency using fast Fourier transformation

Grant,

Larken,

Reitemeier

et al. 2023

Preprint

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Cilia are motile hair-like structures that play a vital role in our body. Accurate assessment of ciliary beat frequency is pivotal for investigating ciliary dynamics and diagnosing ciliopathies. This study aims to develop software for accurately measuring the beat frequency of cilia captured using high-speed video microscopy.To achieve this, we developed theciliRpackage in R, which was validated against manual counting and three other automated methods of counting cilia beat frequency. The results showed thatciliRproduced results that were comparable to manual counting. The accuracy ofciliRwas defined by its ability to reduce noise, including only counting data in a biologically significant range (0-60 Hz).Our software is a valuable tool for researchers in the field of ciliobiology as it offers a reliable method for detailed ciliary function analysis, thereby contributing to the broader understanding of mechanisms underlying ciliary-related disease.We encourage researchers to try this package and feed-back their findings to the authors. Instructions for use and processes for providing feedback are provided in supplementary material.SummaryciliR is a novel R package designed for analysing ciliary beat frequency (CBF) via ImageJ and RStudio. The advantage of the ciliR system, lies in its integration with the R environment, increasing processing speed and access to data visualization tools and analysis pipelines available in other R packages. The open-source platform invites community feedback to refine functionality, aiming to advance ciliopathy research with an accessible, comprehensive toolkit.

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Methods for the assessment of human airway ciliary function

Cited by 7 publications

References 72 publications

A novel in vitro tubular model to recapitulate features of distal airways: The bronchioid

A novel in vitro tubular model to recapitulate features of distal airways: The bronchioid

The RSPH4A Gene in Primary Ciliary Dyskinesia

ciliR: an R package for determining ciliary beat frequency using fast Fourier transformation

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