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INTESTINAL Publication-derived

Cystic Fibrosis Colonic Organoids

Full SOP with Academic or a plan

Source Dekkers et al., 2016 · Wilhelmina Children's Hospital, University Medical Center Utrecht · 10.1126/scitranslmed.aad8278

Authors: Johanna F. Dekkers, Gitte Berkers, Evelien Kruisselbrink, Annelotte Vonk, Hugo R. de Jonge, Hettie M. Janssens, Inez Bronsveld, Eduard A. van de Graaf, Edward E. S. Nieuwenhuis, Roderick H. J. Houwen, Frank P. Vleggaar, Johanna C. Escher, Yolanda B. de Rijke, Christof J. Majoor, Harry G. M. Heijerman, Karin M. de Winter–de Groot, Hans Clevers, Cornelis K. van der Ent, Jeffrey M. Beekman Duration: 10 days 4 phases Cell source: Patient-Derived Primary Rectal Epithelial Cells (Cystic Fibrosis)

Abstract

This protocol establishes rectal organoid cultures from CF patients and healthy controls to measure residual CFTR function and responses to CFTR-modulating drugs (VX-809 and VX-770) using two functional assays: forskolininduced swelling (FIS) and steady-state lumen area (SLA). The protocol enables preclinical identification of drug-responsive CF patients, particularly those with rare CFTR mutations.

Cell source
Patient-Derived Primary Rectal Epithelial Cells (Cystic Fibrosis)
Application
Disease modeling; Drug screening for CFTR-modulating compounds

Protocol overview

23 steps across 4 phases

Phase 1 · Crypt Isolation and Organoid Establishment Day 0-10
  1. a Obtain rectal suction biopsy
  2. b Isolate crypts from biopsy tissue
  3. c Seed crypts in Matrigel
  4. d Prepare and add complete growth medium
  5. e Maintain and passage organoid cultures
Phase 2 · Forskolin-Induced Swelling (FIS) Assay Day 7-10 (assay performed on 7-10 day old cultures)
  1. a Prepare organoids for assay
  2. b Incubate organoids with calcein green
  3. c Stimulate organoids with forskolin and drug
  4. d Image organoids by confocal live cell microscopy
  5. e Quantify organoid swelling by image analysis
  6. f Calculate area under the curve (AUC)
Phase 3 · Steady-State Lumen Area (SLA) Assay Day 7-10 (assay performed on 7-10 day old cultures)
  1. a Seed organoids for SLA assay
  2. b Incubate organoids with drug treatments for 24 hours
  3. c Label organoids with calcein green
  4. d Acquire confocal images
  5. e Quantify total and luminal organoid area
  6. f Calculate steady-state lumen area (SLA) percentage
Phase 4 · Clinical Validation (Optional: In Vivo Assessment) Variable (4 weeks treatment + 4 weeks washout)
  1. a Select candidates based on in vitro organoid response
  2. b Administer CFTR-modulating drug
  3. c Measure CFTR-dependent clinical markers during treatment
  4. d Implement washout period
  5. e Measure clinical parameters after washout
  6. f Correlate in vitro and in vivo responses

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Attribution

This SOP was written by Organthis based on the published method in Dekkers et al., 2016. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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