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Nasospheroids permit measurements of CFTR-dependent fluid transport
Jennifer S. Guimbellot, … , Ilona Jaspers, Martina Gentzsch
Jennifer S. Guimbellot, … , Ilona Jaspers, Martina Gentzsch
Published November 16, 2017
Citation Information: JCI Insight. 2017;2(22):e95734. https://doi.org/10.1172/jci.insight.95734.
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Research Article Cell biology Pulmonology

Nasospheroids permit measurements of CFTR-dependent fluid transport

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Abstract

Expansion of novel therapeutics to all patients with cystic fibrosis (CF) requires personalized CFTR modulator therapy. We have developed nasospheroids, a primary cell culture–based model derived from individual CF patients and healthy subjects by a minimally invasive nasal biopsy. Confocal microscopy was utilized to measure CFTR activity by analyzing changes in cross-sectional area over time that resulted from CFTR-mediated ion and fluid movement. Both the rate of change over time and AUC were calculated. Non-CF nasospheroids with active CFTR-mediated ion and fluid movement showed a reduction in cross-sectional area, whereas no changes were observed in CF spheroids. Non-CF spheroids treated with CFTR inhibitor lost responsiveness for CFTR activation. However, nasospheroids from F508del CF homozygotes that were treated with lumacaftor and ivacaftor showed a significant reduction in cross-sectional area, indicating pharmacologic rescue of CFTR function. This model employs a simple measurement of size corresponding to changes in CFTR activity and is applicable for detection of small changes in CFTR activity from individual patients in vitro. Advancements of this technique will provide a robust model for individualized prediction of CFTR modulator efficacy.

Authors

Jennifer S. Guimbellot, Justin M. Leach, Imron G. Chaudhry, Nancy L. Quinney, Susan E. Boyles, Michael Chua, Inmaculada Aban, Ilona Jaspers, Martina Gentzsch

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Figure 1

Nasospheroids are highly differentiated, hollow spheres with the cilia oriented to the media bath.

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Nasospheroids are highly differentiated, hollow spheres with the cilia o...
(A) Confocal fluorescent microscopy of living nasospheroids treated with calcein green (cytoplasm of living cells), plasma membrane orange (cilia and plasma membranes), and DRAQ5 (1, 5-bis[(2-[di-methylamino]ethyl)amino]-4, 8-dihydroxyanthracene-9, 10-dione, nuclei, in blue). Scale bar: 100 μM. (B) Nasospheroids were fixed in 4% paraformaldehyde and embedded in hydroxyethyl agarose, followed by paraffin-embedding and cryosection to produce thin sections. Processing results in dehydration and reduction in overall size. These sections were stained with H&E as described in Methods and show intact epithelial layer with ciliated and nonciliated cells. Scale bar: 10 μM. (C) Intact nasospheroids were fixed in ice-cold methanol followed by incubation with anti-CFTR antibody and fluorescent-tagged secondary antibody as described in Methods. Nuclei are stained blue with DAPI. Scale bar: 50 μM. (D) Illustration showing expected flow of anions and water with CFTR activation.

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