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Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia

BACKGROUND: Three-dimensional explant spheroid formation is an ex vivo technique previously used in studies of airway epithelial ion and water transport. Explanted cells and sheets of nasal epithelium form fully differentiated spheroids enclosing a partly fluid-filled lumen with the ciliated apical...

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Autores principales: Marthin, June Kehlet, Stevens, Elizabeth Munkebjerg, Larsen, Lars Allan, Christensen, Søren Tvorup, Nielsen, Kim Gjerum
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364668/
https://www.ncbi.nlm.nih.gov/pubmed/28344781
http://dx.doi.org/10.1186/s13630-017-0049-5
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author Marthin, June Kehlet
Stevens, Elizabeth Munkebjerg
Larsen, Lars Allan
Christensen, Søren Tvorup
Nielsen, Kim Gjerum
author_facet Marthin, June Kehlet
Stevens, Elizabeth Munkebjerg
Larsen, Lars Allan
Christensen, Søren Tvorup
Nielsen, Kim Gjerum
author_sort Marthin, June Kehlet
collection PubMed
description BACKGROUND: Three-dimensional explant spheroid formation is an ex vivo technique previously used in studies of airway epithelial ion and water transport. Explanted cells and sheets of nasal epithelium form fully differentiated spheroids enclosing a partly fluid-filled lumen with the ciliated apical surface facing the outside and accessible for analysis of ciliary function. METHODS: We performed a two-group comparison study of ciliary beat pattern and ciliary beat frequency in spheroids derived from nasal airway epithelium in patients with primary ciliary dyskinesia (PCD) and in healthy controls. Nasal ciliary cells and sheets were removed on day 1 by nasal brush biopsy and analyzed with regard to ciliary beat pattern—and frequency using high-speed video imaging for standard reference values. Three-dimensional explant spheroid formation was initiated in the same individual on the same day by incubation of cells and sheets from a separate brush biopsy. Harvested spheroids were analyzed earliest possible and values of spheroid ciliary beat pattern and frequency were compared to the corresponding reference values from day 1. RESULTS: Spheroids formed fast in serum-free culture medium. Formation was successful in 15 out of 18 (82%) sampled individuals. Thus, formation was successful in seven healthy controls and eight PCD patients, while unsuccessful in 3 with PCD due to infection. Median (range) number of days in culture before harvesting of spheroids was 4 (1–5) in healthy versus 2 (1–5) in PCD. Spheroid ciliary beat pattern and frequency were unchanged compared to their corresponding day 1 standard reference values. Spheroid ciliary beat frequency discriminated highly significant between healthy controls (9.3 Hz) and PCD patients (2.4 Hz) (P < 0.0001). Survival of spheroids was 16 days in a single healthy person. CONCLUSION: Patient-specific three-dimensional explant spheroid formation from a minimal invasive nasal brush biopsy is a feasible, fast and valid ex vivo method to assess ciliary function with potential of aiding the diagnosis of PCD. In addition, it may be a useful model in the investigation of pathophysiological aspects and drug effects in human nasal airway epithelium. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13630-017-0049-5) contains supplementary material, which is available to authorized users.
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spelling pubmed-53646682017-03-24 Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia Marthin, June Kehlet Stevens, Elizabeth Munkebjerg Larsen, Lars Allan Christensen, Søren Tvorup Nielsen, Kim Gjerum Cilia Research BACKGROUND: Three-dimensional explant spheroid formation is an ex vivo technique previously used in studies of airway epithelial ion and water transport. Explanted cells and sheets of nasal epithelium form fully differentiated spheroids enclosing a partly fluid-filled lumen with the ciliated apical surface facing the outside and accessible for analysis of ciliary function. METHODS: We performed a two-group comparison study of ciliary beat pattern and ciliary beat frequency in spheroids derived from nasal airway epithelium in patients with primary ciliary dyskinesia (PCD) and in healthy controls. Nasal ciliary cells and sheets were removed on day 1 by nasal brush biopsy and analyzed with regard to ciliary beat pattern—and frequency using high-speed video imaging for standard reference values. Three-dimensional explant spheroid formation was initiated in the same individual on the same day by incubation of cells and sheets from a separate brush biopsy. Harvested spheroids were analyzed earliest possible and values of spheroid ciliary beat pattern and frequency were compared to the corresponding reference values from day 1. RESULTS: Spheroids formed fast in serum-free culture medium. Formation was successful in 15 out of 18 (82%) sampled individuals. Thus, formation was successful in seven healthy controls and eight PCD patients, while unsuccessful in 3 with PCD due to infection. Median (range) number of days in culture before harvesting of spheroids was 4 (1–5) in healthy versus 2 (1–5) in PCD. Spheroid ciliary beat pattern and frequency were unchanged compared to their corresponding day 1 standard reference values. Spheroid ciliary beat frequency discriminated highly significant between healthy controls (9.3 Hz) and PCD patients (2.4 Hz) (P < 0.0001). Survival of spheroids was 16 days in a single healthy person. CONCLUSION: Patient-specific three-dimensional explant spheroid formation from a minimal invasive nasal brush biopsy is a feasible, fast and valid ex vivo method to assess ciliary function with potential of aiding the diagnosis of PCD. In addition, it may be a useful model in the investigation of pathophysiological aspects and drug effects in human nasal airway epithelium. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13630-017-0049-5) contains supplementary material, which is available to authorized users. BioMed Central 2017-03-23 /pmc/articles/PMC5364668/ /pubmed/28344781 http://dx.doi.org/10.1186/s13630-017-0049-5 Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Marthin, June Kehlet
Stevens, Elizabeth Munkebjerg
Larsen, Lars Allan
Christensen, Søren Tvorup
Nielsen, Kim Gjerum
Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia
title Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia
title_full Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia
title_fullStr Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia
title_full_unstemmed Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia
title_short Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia
title_sort patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364668/
https://www.ncbi.nlm.nih.gov/pubmed/28344781
http://dx.doi.org/10.1186/s13630-017-0049-5
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