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GASTRIC Publication-derived

Rectal Organoid Establishment and Forskolin-Induced Swelling Assay for CFTR Modulator Drug Response Testing in Cystic Fibrosis

Source Berkers et al., 2019 · University Medical Center Utrecht, Department of Pediatric Pulmonology · 10.1016/j.celrep.2019.01.068

👤 Gitte Berkers, Peter van Mourik, Annelotte M. Vonk, Evelien Kruisselbrink, Johanna F. Dekkers, Karin M. de Winter-de Groot, Hubertus G.M. Arets, Jeffrey M. Beekman, Cornelis K. van der Ent ⏱ 180 days 📋 4 phases 🧫 Patient-Derived Rectal Epithelial Organoids from CF Patients

Abstract

This protocol describes the culture of rectal organoids derived from cystic fibrosis patients and the functional assessment of CFTR modulator drug responses using forskolin-induced swelling (FIS) measurements. The assay quantifies individual drug efficacy in vitro and correlates with clinical outcomes (ppFEV1 and sweat chloride concentration), enabling personalized treatment selection for CF patients.

Cell source
Patient-Derived Rectal Epithelial Organoids from CF Patients
Application
Disease modeling and personalized drug screening for cystic fibrosis treatment selection

Protocol overview

19 steps across 4 phases

Rectal Organoid Culture and Maintenance Day 1 to Day 180 (6 months storage)
  1. 1 Obtain rectal tissue biopsy
  2. 2 Establish organoid culture from rectal tissue
  3. 3 Maintain organoid cultures in growth medium
Preparation for Forskolin-Induced Swelling Assay Day of assay (pre-experiment setup)
  1. 1 Prepare test drugs and control
  2. 2 Prepare forskolin working solutions
  3. 3 Prepare organoid wells for imaging
  4. 4 Prepare fluorescent labeling solution
Forskolin-Induced Swelling Assay Execution Single assay day
  1. 1 Label organoids with Calcein, AM
  2. 2 Establish baseline organoid area (t=0 min)
  3. 3 Add DMSO control or drug + forskolin
  4. 4 Acquire real-time confocal images for 60 minutes
  5. 5 Measure organoid area at end-point (t=60 min)
  6. 6 Quantify organoid swelling response
Data Analysis and Clinical Correlation Post-assay analysis (variable timeline)
  1. 1 Calculate drug response for each sample
  2. 2 Collect in vivo clinical endpoint data
  3. 3 Perform Pearson correlation analysis
  4. 4 Generate receiver operating characteristic (ROC) curves
  5. 5 Calculate predictive values
  6. 6 Perform leave-one-out cross-validation

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Berkers et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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