title: Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients.
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 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.
authors.affiliation: Foundation Hubrecht Organoid Technology.
authors.forename: Sylvia F
authors.initials: SF
authors.lastname: Boj
authors.affiliation: Department of Pediatric Pulmonology, Regenerative Medicine Centre Utrecht, Wilhelmina Children's Hospital, University Medical Centre Utrecht.
authors.forename: Annelotte M
authors.initials: AM
authors.lastname: Vonk
authors.affiliation: Foundation Hubrecht Organoid Technology.
authors.forename: Marvin
authors.initials: M
authors.lastname: Statia
authors.affiliation: Foundation Hubrecht Organoid Technology.
authors.forename: Jinyi
authors.initials: J
authors.lastname: Su
authors.affiliation: Foundation Hubrecht Organoid Technology.
authors.forename: Robert R G
authors.initials: RR
authors.lastname: Vries
authors.affiliation: Department of Pediatric Pulmonology, Regenerative Medicine Centre Utrecht, Wilhelmina Children's Hospital, University Medical Centre Utrecht; j.beekman@umcutrecht.nl.
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Beekman
authors.affiliation: Foundation Hubrecht Organoid Technology; Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht; h.clevers@hubrecht.eu.
authors.forename: Hans
authors.initials: H
authors.lastname: Clevers
mesh_terms: D001681:Biological Assay / Q000379:methods; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism; D017136:Ion Transport; D009154:Mutation; D009940:Organoids / Q000187:drug effects* / Q000378:metabolism; D014665:Vasodilator Agents / Q000494:pharmacology*
chemical_list: C505032:CFTR protein, human; D014665:Vasodilator Agents; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D000242:Cyclic AMP