Protocol for Application, Standardization and Validation of the Forskolin-Induced Swelling Assay in Cystic Fibrosis Human Colon Organoids

Annelotte M Vonk; Peter van Mourik; Anabela S Ramalho; Iris A L Silva; Marvin Statia; Evelien Kruisselbrink; Sylvia W F Suen; Johanna F Dekkers; Frank P Vleggaar; Roderick H J Houwen; Jasper Mullenders; Sylvia F Boj; Robert Vries; Margarida D Amaral; Kris de Boeck; Cornelis K van der Ent; Jeffrey M Beekman

doi:10.1016/j.xpro.2020.100019

Protocol for Application, Standardization and Validation of the Forskolin-Induced Swelling Assay in Cystic Fibrosis Human Colon Organoids

STAR Protoc. 2020 Jun 3;1(1):100019. doi: 10.1016/j.xpro.2020.100019. eCollection 2020 Jun 19.

Authors

Annelotte M Vonk^{1

2}, Peter van Mourik^{1

2}, Anabela S Ramalho³, Iris A L Silva⁴, Marvin Statia⁵, Evelien Kruisselbrink^{1

2}, Sylvia W F Suen^{1

2}, Johanna F Dekkers⁶, Frank P Vleggaar⁷, Roderick H J Houwen⁸, Jasper Mullenders⁵, Sylvia F Boj⁵, Robert Vries⁵, Margarida D Amaral⁴, Kris de Boeck³, Cornelis K van der Ent¹, Jeffrey M Beekman^{1

2}

Affiliations

¹ Dept. of Pediatric Pulmonology, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands.
² Regenerative Medicine Utrecht, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands.
³ Katholieke Universiteit Leuven, Dept. of Development and Regeneration, Leuven, Belgium.
⁴ University of Lisboa, Faculty of Sciences, BioISI- Biosystems & Integrative Sciences Institute, Lisboa, Portugal.
⁵ Hubrecht Organoid Technology (HUB), Utrecht, the Netherlands.
⁶ Prinses Maxima Centrum, Utrecht, the Netherlands.
⁷ Gastroenterology and Hepatology, University Medical Center Utrecht, Utrecht, the Netherlands.
⁸ Dept. of Pediatric Gastroenterology, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, the Netherlands.

Abstract

This protocol describes the isolation, handling, culture of, and experiments with human colon stem cell organoids in the context of cystic fibrosis (CF). In human colon organoids, the function of cystic fibrosis transmembrane conductance regulator (CFTR) protein and its rescue by CFTR modulators can be quantified using the forskolin-induced swelling assay. Implementation procedures and validation experiments are described for six CF human colon organoid lines, and representative CFTR genotypes are tested for basal CFTR function and response to CFTR-modulating drugs. For complete details on the use and execution of this protocol, please refer to Dekkers et al (2016) and Berkers and van Mourik (2019).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biological Assay / methods*
Cells, Cultured
Colforsin / pharmacology*
Colon* / drug effects
Colon* / metabolism
Cystic Fibrosis / metabolism*
Edema
Humans
Organoids* / drug effects
Organoids* / metabolism
Reproducibility of Results

Substances

Colforsin