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INTESTINAL Publication-derived

Functional CFTR Assay Using Primary Cystic Fibrosis Intestinal Organoids

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

👤 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 ⏱ 24 days 📋 7 phases 🧫 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

Organoid Culture Establishment Days 1–10
  1. 1 Biopsy collection and crypt isolation
  2. 2 Matrigel embedding and seeding
  3. 3 Culture medium preparation and initial culture
  4. 4 Media refreshment and passaging
Organoid Seeding and Labeling Days 7–8
  1. 5 Organoid transfer to 96-well plate
  2. 6 Calcein green labeling
CFTR Inhibitor Pretreatment (if applicable) Day 8 (or experimental day)
  1. 7 Optional: CFTR inhibition pretreatment
CFTR Correction or Potentiation Pretreatment (if applicable) 24 hours prior to stimulation assay
  1. 8 Temperature rescue of F508del CFTR (optional)
  2. 9 Chemical corrector pretreatment
Forskolin Stimulation and Live-Cell Confocal Imaging Day 8 (or experimental endpoint)
  1. 10 Forskolin stimulation setup
  2. 11 Confocal live-cell microscopy acquisition
  3. 12 Image analysis and quantification
  4. 13 Area under the curve (AUC) calculation
Supporting Molecular Characterization (Optional but Recommended) Variable (in parallel or after stimulation assay)
  1. 14 Western blot analysis of CFTR protein expression
  2. 15 CFTR deglycosylation and characterization
  3. 16 Immunofluorescence microscopy for CFTR localization
  4. 17 Quantitative RT-PCR for CFTR mRNA expression
Optional Validation: Ex Vivo Intestinal Current Measurement (ICM) Concurrent or prior to organoid studies
  1. 18 Rectal biopsy collection and ICM setup
  2. 19 Sequential compound addition and current measurement
  3. 20 Comparison of ICM and FIS data

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Dekkers et al., 2013. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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