Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Nov 16;2(22):e95734.
doi: 10.1172/jci.insight.95734.

Nasospheroids Permit Measurements of CFTR-dependent Fluid Transport

Affiliations
Free PMC article

Nasospheroids Permit Measurements of CFTR-dependent Fluid Transport

Jennifer S Guimbellot et al. JCI Insight. .
Free PMC article

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.

Keywords: Cell Biology; Epithelial transport of ions and water; Genetic diseases; Pulmonology.

Conflict of interest statement

Conflict of interest: M. Gentzsch, J.S. Guimbellot, N.L. Quinney, and S.E. Boyles are an inventors of the technology “airway sphere cultures as diagnostic device to monitor pharmacological responses of ion channels,” which is licensed to Path BioAnalytics. M. Gentzsch, J.S. Guimbellot, N.L. Quinney, and S.E. Boyles could receive royalties related to the license in the future. These relationships have been disclosed to and are under management by the University of North Carolina at Chapel Hill.

Figures

Figure 1
Figure 1. Nasospheroids are highly differentiated, hollow spheres with the cilia oriented to the media bath.
(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.
Figure 2
Figure 2. Non-CF nasospheroids show reduction in cross-sectional area over time when CFTR is stimulated by forskolin.
(A) Representative non–cystic fibrosis (CF) nasospheroids are stained with calcein green. Z-stacks are taken sequentially using confocal microscopy and the maximal projection generated. The same nasospheroids are shown at 30 and 75 minutes. Scale bars: 50 μM. (B) The starting size of each nasospheroid was set at 1, and the fractional reduction of each was calculated at all time points. The average fractional reduction was plotted at each time point. Stimulated nasospheroids were treated with 10 μM forskolin, amiloride, and IBMX. Nonstimulated spheroids were treated with an equal concentration of vehicle dimethyl sulfoxide (DMSO). Stimulated spheroids + inhibitor were treated with CFTRinh-172 at time of testing. Analysis of variance was calculated for the groups. Mean values ± SD are shown.*P < 0.00001 and ‡P < 0.001 for 1-way ANOVA and paired t tests between time points. (C) Each nasospheroid absolute size of maximal projection at each time point was plotted over time for all time points in the “Raw” graph. The “Fitted” graph represents the fitted slopes for each nasospheroid after linear regression (see Methods). (D) Summary data of slopes for all non-CF nasospheroids and non-CF nasospheroids treated with CFTRinh-172. Fitted slopes are represented by a box and dot plot. Each dot is a single nasospheroid. The black box is the mean; the central line is the median; the box borders are the 25th and 75th percentiles. *P < 0.001. (E) Summary data of time-averaged AUC unadjusted for starting size for all non-CF nasospheroids and non-CF nasospheroids treated with CFTRinh-172. AUC is represented by a box plot. *P < 0.0001. n = 9 subjects. A minimum of 3 up to a maximum of 18 nasospheroids were analyzed per subject.
Figure 3
Figure 3. CF nasospheroids show no reduction in cross-sectional area over time when CFTR is stimulated, unless treated with modulators.
(A) Baseline area at start of testing is not significantly different in CF compared with non-CF nasospheroids. (B) The starting size of each nasospheroid was set at 1, and the fractional reduction of each was calculated at all time points. The average fractional reduction was calculated and plotted at each time point. Stimulated nasospheroids were treated with 10 μM forskolin, amiloride, and 3-isobutyl-1-methylxanthine (IBMX). Additional nasospheroids were treated with 5 μM lumacaftor (24 hours) and 1 μM ivacaftor, acute treatment. One-way ANOVA was calculated for the groups, P = 0.047. Paired t test between time 0 and time 75 minutes, *P < 0.0001. (C) Summary data of slopes for all CF nasospheroids represented as a box and dot plot. Each dot is a single nasospheroid. The black box is the mean; the central line is the median; the box borders are the 25th and 75th percentiles. *P <0.0001. (D) Summary data of time-averaged AUC unadjusted for starting size for all CF nasospheroids represented as a box plot.*P = 0.0113. n = 3 subjects. Between 3 and 5 nasospheroids were analyzed per subject.

Similar articles

See all similar articles

Cited by 9 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback