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. 2024 Jan;23(1):169-171.
doi: 10.1016/j.jcf.2023.08.003. Epub 2023 Aug 24.

Pancreatic duct organoid swelling is chloride-dependent

Yunxia O'Malley  1 , Keyan Zarei  2 , Oriana G Calderon Vanegas  1 , Pratibha Singh  1 , T Idil Apak  3 , Mitchell Coleman  4 , Ian M Thornell  5 , Aliye Uc  6
Affiliations

Pancreatic duct organoid swelling is chloride-dependent

Yunxia O'Malley et al. J Cyst Fibros. 2024 Jan.

Abstract

Pancreatic secretions become viscous and acidic in Cystic fibrosis (CF), highlighting the role of CFTR in pancreatic fluid and bicarbonate secretion. Forskolin-induced swelling (FIS) assay developed in intestinal organoids measures residual CFTR function. It is not known whether FIS reflects bicarbonate secretion in pancreas, an organ that secretes near-isotonic NaHCO3 levels. To investigate this, we generated pancreatic duct organoids from CF and non-CF pigs. Epithelial and ductal origin was confirmed with epithelial markers, ion transporters and lack of acinar, islet cell markers. CF organoids were small with no identifiable lumen; CFTR was expressed only in non-CF organoids. Utilizing FIS, organoid size increased only in response to chloride, not bicarbonate. This report highlights pancreatic duct organoids isolated for the first time from CF pigs and evidence for chloride and not bicarbonate driving pancreatic organoid swelling. These organoids would be useful to test chloride permeability of CFTR mutations that cause CF pancreatic disease.

Keywords: Anion secretion; Cystic fibrosis; Duct cells; Pancreas.

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Conflict of interest statement

Declaration of Competing Interest Dr. Uc receives grant support from NIH R01 DK118752 and U01 DK108334. She is a consultant for Cystic Fibrosis Foundation and Abbvie Pharmaceuticals, Inc. Other authors declare no conflicts of interest.

Figures

Figure 1.
Figure 1.. Characterization of Porcine Pancreatic Duct Organoids.
(A, B) Representative bright-field images of organoids 9 days after isolation, at passage 2. Organoids were used for experiments between passages 2 and 5 to avoid any obvious changes in morphology or gene expression. (C, D) Representative immunofluorescent images of organoids, F-actin (red) at apical membrane, Na-K pump (yellow) at basolateral membrane, zona occludens-1 (ZO-1, green) at tight junctions and nuclei (DAPI, blue). (E-I) Agarose gel images showing cDNA samples from organoids, and (J) from whole pancreas extract. No bands were visualized if water was used as substrate (data not shown). Molecular weight (MW) was loaded at both ends of the gel. Data is representative of at least 3 independent experiments. Scale bars are shown.
Figure 2.
Figure 2.. Porcine Pancreatic Duct Organoid Swelling is Chloride Dependent.
(A) Forskolin dose response showing % change in size of non-CF organoids from baseline at 60 minutes in chloride and bicarbonate buffer. (B) Non-CF organoids in various buffers after initial incubation in gluconate buffer, dashed lines denote starting area; open symbols: buffer alone, closed symbols: buffer + forskolin 10 μM. (C) Non-CF and CF organoid swelling utilizing various chloride concentrations at 60 min demonstrating % change from initial size. The curve analysis was done using nonlinear regression curve fit model. (D) Forskolin dose response showing % change in size of CF organoids from baseline at 60 min in chloride and bicarbonate buffer. (E) CF organoids in various buffers after initial incubation in gluconate buffer, open symbols: buffer alone, closed symbols: buffer + forskolin 10 μM. (F) surface area of non-CF and CF organoids. Data represent mean ± STDEV; n=10–15 organoids for each condition, single experiment, repeated 3 times; *p<0.05; **p<0.01; ***p<0.001. One-way ANOVA was used with Dunnett correction for multiple comparisons.
See this image and copyright information in PMC

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