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INTESTINAL Publication-derived

Cystic Fibrosis Intestinal Organoids for Drug Screening

Full SOP with Academic or a plan

Source Dekkers et al., 2013 · Wilhelmina Children's Hospital, University Medical Center, Utrecht, The Netherlands · 10.1038/nm.3201

Authors: Johanna F Dekkers, Caroline L Wigerink, Hugo R de Jonge, Inez Bronsveld, Hettie M Janssens, Karin M de Winter-de Groot, Arianne M Brandsma, Nienke W M de Jong, Marcel J C Bijvelds, Bob J Scholte, Edward E S Nieuwenhuis, Stieneke van den Brink, Hans Clevers, Cornelis K van der Ent, Sabine Middendorp, Jeffrey M Beekman Duration: 24 days 7 phases Cell source: Primary human intestinal epithelial cells (healthy controls and patients with cystic fibrosis); mouse intestinal crypts

Abstract

This protocol describes a quantitative assay for CFTR function in primary mouse and human intestinal organoids using forskolin-induced organoid swelling (FIS). The method enables rapid measurement of fluid secretion capacity—a direct readout of CFTR-mediated anion and fluid transport—and is validated against classical electrophysiological measurements. The assay can detect CFTR dysfunction in cystic fibrosis organoids and restoration of function by temperature rescue or CFTR-targeting drugs, making it suitable for diagnosis, drug screening, and personalized medicine in cystic fibrosis.

Cell source
Primary human intestinal epithelial cells (healthy controls and patients with cystic fibrosis); mouse intestinal crypts
Application
Disease modeling; Drug screening; CFTR functional assessment

Protocol overview

20 steps across 7 phases

Phase 1 · Organoid Culture Establishment Days 1-10
  1. a Biopsy collection and crypt isolation
  2. b Matrigel embedding and seeding
  3. c Culture medium preparation and initial culture
  4. d Media refreshment and passaging
Phase 2 · Organoid Seeding and Labeling Days 7-8
  1. a Organoid transfer to 96-well plate
  2. b Calcein green labeling
Phase 3 · CFTR Inhibitor Pretreatment (if applicable) Day 8 (or experimental day)
  1. a Optional: CFTR inhibition pretreatment
Phase 4 · CFTR Correction or Potentiation Pretreatment (if applicable) 24 hours prior to stimulation assay
  1. a Temperature rescue of F508del CFTR (optional)
  2. b Chemical corrector pretreatment
Phase 5 · Forskolin Stimulation and Live-Cell Confocal Imaging Day 8 (or experimental endpoint)
  1. a Forskolin stimulation setup
  2. b Confocal live-cell microscopy acquisition
  3. c Image analysis and quantification
  4. d Area under the curve (AUC) calculation
Phase 6 · Supporting Molecular Characterization (Optional but Recommended) Variable (in parallel or after stimulation assay)
  1. a Western blot analysis of CFTR protein expression
  2. b CFTR deglycosylation and characterization
  3. c Immunofluorescence microscopy for CFTR localization
  4. d Quantitative RT-PCR for CFTR mRNA expression
Phase 7 · Optional Validation: Ex Vivo Intestinal Current Measurement (ICM) Concurrent or prior to organoid studies
  1. a Rectal biopsy collection and ICM setup
  2. b Sequential compound addition and current measurement
  3. c Comparison of ICM and FIS data

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Attribution

This SOP was written by Organthis based on the published method in Dekkers et al., 2013. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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