Protocol for Application, Standardization and Validation of the Forskolin-Induced Swelling Assay in Cystic Fibrosis Human Colon Organoids
Vonk, Annelotte M; van Mourik, Peter; Ramalho, Anabela S; Silva, Iris A L; Statia, Marvin; Kruisselbrink, Evelien; Suen, Sylvia W F; Dekkers, Johanna F; Vleggaar, Frank P; Houwen, Roderick H J; Mullenders, Jasper; Boj, Sylvia F; Vries, Robert; Amaral, Margarida D; de Boeck, Kris; van der Ent, Cornelis K; Beekman, Jeffrey M
(2020) STAR protocols, volume 1, issue 1, pp. 1 - 31
(Article)
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
... read more
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).
show less
Download/Full Text
Keywords: General Biochemistry,Genetics and Molecular Biology, General Neuroscience, General Immunology and Microbiology, Journal Article
ISSN: 2666-1667
Publisher: Cell Press
Note: Funding Information: This project has received funding from the European Union's Horizon 2020 research and innovation program under grant proposal number 755021-2. This project has been supported by the Dutch Cystic Fibrosis Foundation (NCFS). These protocols have been endorsed by the European CF Society (ECFS) and by the European Reference Network for Respiratory Diseases (ERN-LUNG) in 2019 and aim to standardize methods to ensure high reproducibility and correct interpretation of results among different laboratories. Conceptualization: A.M.V. P.M. C.K.E. J.M.B.; Methodology, A.M.V. S.W.F.S. M.S. F.P.V. R.H.J.H. E.K. J.F.D. S.F.B. J.M.; Validation; A.M.V. A.S.R. I.A.L.S. S.W.F.S. J.F.D.; Investigation, A.M.V. A.S.R. I.A.L.S. S.W.F.S.; Writing ? Original Draft; A.M.V. P.M. M.S. A.S.R. I.A.L.S. M.S.; Writing ? Review & Editing, J.M. S.F.B. R.V. M.D.A. K.B. J.M.B.; Funding Acquisition, P.M. J.M.B. C.K.E.; Supervision, C.K.E., J.M.B. J.F.D. is an inventor on a patent related to the organoid technology. M.A.A. reports grants from Gilead Pharmaceuticals, Vertex Pharmceuticals, Proteostasis Therapeutics and personal fees from Vertex Pharmceuticals and PTC Pharmaceuticals outside the submitted work. K.B. reports consultancy work for CHIESI, grants from VERTEX, advisory board meetings for PROTEOSTASIS and ELOXX, grants from and advisory board meetings for GALAPAGOS, and advisory board meetings for TRANSLATE BIO outside the submitted work. R.V. is CEO of Hubrecht Organoid Technology, which provides organoid services to companies. C.K.E. reports grants from GSK, Nutricia, TEVA, Gilead, Vertex, ProQR, Proteostasis, Galapagos NV, and Eloxx outside the submitted work and has a patent 10006904 with royalties paid. J.M.B. is inventor on a patent related to CFTR function measurements in organoids and has received financial royalties. He also participates in industry-sponsored clinical trials (Galapagos NV, Vertex Pharmaceuticals, Proteostasis, Eloxx Pharmaceuticals) and has received travel reimbursements and speaker's fees. The other authors have nothing to disclose. Funding Information: J.F.D. is an inventor on a patent related to the organoid technology. M.A.A. reports grants from Gilead Pharmaceuticals, Vertex Pharmceuticals, Proteostasis Therapeutics and personal fees from Vertex Pharmceuticals and PTC Pharmaceuticals outside the submitted work. K.B. reports consultancy work for CHIESI, grants from VERTEX, advisory board meetings for PROTEOSTASIS and ELOXX, grants from and advisory board meetings for GALAPAGOS, and advisory board meetings for TRANSLATE BIO outside the submitted work. R.V. is CEO of Hubrecht Organoid Technology, which provides organoid services to companies. C.K.E. reports grants from GSK, Nutricia, TEVA, Gilead, Vertex, ProQR, Proteostasis, Galapagos NV, and Eloxx outside the submitted work and has a patent 10006904 with royalties paid. J.M.B. is inventor on a patent related to CFTR function measurements in organoids and has received financial royalties. He also participates in industry-sponsored clinical trials (Galapagos NV, Vertex Pharmaceuticals, Proteostasis, Eloxx Pharmaceuticals) and has received travel reimbursements and speaker’s fees. The other authors have nothing to disclose. Funding Information: This project has received funding from the European Union's Horizon 2020 research and innovation program under grant proposal number 755021-2 . This project has been supported by the Dutch Cystic Fibrosis Foundation (NCFS). These protocols have been endorsed by the European CF Society (ECFS) and by the European Reference Network for Respiratory Diseases (ERN-LUNG) in 2019 and aim to standardize methods to ensure high reproducibility and correct interpretation of results among different laboratories. Publisher Copyright: © 2020 The Authors
(Peer reviewed)
