Repeatability and reproducibility of the Forskolin-induced swelling (FIS) assay on intestinal organoids from people with Cystic Fibrosis

Marlou C Bierlaagh; Anabela S Ramalho; Iris A L Silva; Annelotte M Vonk; Rutger M van den Bor; Peter van Mourik; Johanna Pott; Sylvia W F Suen; Sylvia F Boj; Robert G J Vries; Elise Lammertyn; François Vermeulen; Margarida D Amaral; Kris de Boeck; Cornelis K van der Ent; Jeffrey M Beekman

doi:10.1016/j.jcf.2024.04.014

Repeatability and reproducibility of the Forskolin-induced swelling (FIS) assay on intestinal organoids from people with Cystic Fibrosis

J Cyst Fibros. 2024 May 14:S1569-1993(24)00060-2. doi: 10.1016/j.jcf.2024.04.014. Online ahead of print.

Authors

Marlou C Bierlaagh¹, Anabela S Ramalho², Iris A L Silva³, Annelotte M Vonk¹, Rutger M van den Bor⁴, Peter van Mourik¹, Johanna Pott⁵, Sylvia W F Suen¹, Sylvia F Boj⁵, Robert G J Vries⁵, Elise Lammertyn⁶, François Vermeulen², Margarida D Amaral³, Kris de Boeck², Cornelis K van der Ent⁷, Jeffrey M Beekman⁸

Affiliations

¹ Dept. of Pediatric Pulmonology, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands; Regenerative Medicine Center Utrecht, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
² KULeuven, Dept. of Development and Regeneration, Leuven, Belgium.
³ BioISI- Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisboa, Lisboa, Portugal.
⁴ Dept. of Data Science and Biostatistics, Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands.
⁵ HUB Organoids B.V. (HUB), Utrecht, The Netherlands.
⁶ Cystic Fibrosis Europe & the Belgian Cystic Fibrosis Association, Brussels, Belgium.
⁷ Regenerative Medicine Center Utrecht, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
⁸ Dept. of Pediatric Pulmonology, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands; Regenerative Medicine Center Utrecht, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands; Centre for Living Technologies, Alliance Eindhoven University of Technology, Wageningen University & Research, Utrecht University and University Medical Center Utrecht (EWUU), Utrecht, The Netherlands. Electronic address: J.Beekman@umcutrecht.nl.

PMID: 38749892
DOI: 10.1016/j.jcf.2024.04.014

Abstract

Background: The forskolin-induced swelling (FIS) assay measures CFTR function on patient-derived intestinal organoids (PDIOs) and may guide treatment selection for individuals with Cystic Fibrosis (CF). The aim of this study is to demonstrate the repeatability and reproducibility of the FIS assay following a detailed Standard Operating Procedure (SOP), thus advancing the validation of the assay for precision medicine (theranostic) applications.

Methods: Over a 2-year period, FIS responses to CFTR modulators were measured in four European labs. PDIOs from six subjects with CF carrying different CFTR genotypes were used to assess the repeatability and reproducibility across the dynamic range of the assay.

Results: Technical, intra-assay repeatability was high (Lin's concordance correlation coefficient (CCC) 0.95-0.98). Experimental, within-subject repeatability was also high within each lab (CCCs all >0.9). Longer-term repeatability (>1 year) showed more variability (CCCs from 0.67 to 0.95). The reproducibility between labs was also high (CCC ranging from 0.92 to 0.97). Exploratory analysis also found that between-lab percentage of agreement of dichotomized CFTR modulator outcomes for predefined FIS thresholds ranged between 78 and 100 %.

Conclusions: The observed repeatability and reproducibility of the FIS assay within and across different labs is high and support the use of FIS as biomarker of CFTR function in the presence or absence of CFTR modulators.

Keywords: Cystic fibrosis; Forskolin-induced swelling assay; Patient-derived intestinal organoids; Precision medicine; Repeatability; Reproducibility.