Forskolin-induced swelling in intestinal organoids: An in vitro assay for assessing drug response in cystic fibrosis patients
Boj, Sylvia F.; Vonk, Annelotte M; Statia, Marvin; Su, Jinyi; Vries, Robert R G; Beekman, Jeffrey M.; Clevers, Hans
(2017) Jove-Journal of visualized experiments, volume 2017, issue 120
(Article)
Abstract
Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that
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contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 °C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.
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Keywords: Biopsy, CFTR function, Cystic fibrosis, Forskolin, Human colorectal organoids, Issue 120, Ivacaftor (VX-770), Lumacaftor (VX-809), Medicine, Organoid swelling, Primary cell culture, General Neuroscience, General Chemical Engineering, General Immunology and Microbiology, General Biochemistry,Genetics and Molecular Biology
ISSN: 1940-087X
Publisher: MYJoVE Corporation
Note: Funding Information: This work was supported by the HIT-CF program of the Dutch CF foundation (NCFS), ZonMW (40-00812-98-14103), the Wilhelmina Children?s Hospital Research Fund and CZ, and Zilverenkruis/Achmea. We would like to thank S. Heida-Michel, M. Geerdink, K.M. de Winter-de Groot, and G. Berkers (Department of Pediatric Pulmonology, Wilhelmina Children's Hospital, UMC Utrecht), and R.H.J. Houwen (Department of Pediatric Gastroenterology, Wilhelmina Children's Hospital, UMC Utrecht) for approaching the patients and getting the biopsies for the generation of a CF Biobank. Publisher Copyright: © 2017 Journal of Visualized Experiments.
(Peer reviewed)