Peter van Mourik

105 Forskolin-induced swelling protocol European FIS validation & standardization protocol Timing ~ 8 weeks To ensure correct implementation of the human organoid technology and the application in the FIS assay, six reference human colon organoid lines (covering different classes of CFTR mutations from severe to mild -see table 5). These organoids lines were tested for basal CFTR function and specific drug response to available CFTR modulators (VX-661 and VX-770). Based on the following protocol, swelling data (technical duplicates at three time points with weekly intervals (n=3)) were generated to compare the qualitative and quantitative results of the implemented organoid technology and FIS assay (not yet published data). These reference organoid lines were selected based on prevalent CFTR genotypes (see table 5) and differences in CFTR basal function and response to CFTR modulators. Researchers that follow the detailed work instructions presented in this protocol should find identical or highly reproducible results (within 10% of AUC values, not yet published data) for these reference lines to confirm successful implementation of the human intestinal organoid technology and FIS assay. Table 5. Overview of the different organoid cell lines that can be used to validate FIS assay results. Validation Organoid Cell-lines CF class mutation Specific genotype HUB ID code Class I/ Class I G542X / G542X HUB-02-D2-121 Class II/ Class I F508del / R1162X HUB-02-D2-038 Class II/ Class II F508del/ F508del HUB-02-D2-341 Class II/ Class III F508del/ G551D HUB-02-D2-043 Class II/ Class III F508del/S1251N HUB-02-D2-103 Class II/ Class IV (mild phenotype) F508del / R117H - 7T HUB-02-D2-004 A. Preparations for validation protocol Medium amounts: 1 liter CM +/+ (see ‘Stock preparation of human colon organoid medium components’ for instructions on media preparation) 500 mL 2x CM -/- Prepare 10 x 100 mL bottles with 50 mL stock and store at -20 °C 500 mL WCM Prepare 2 batches of > 375 mL (> 20 x 145 mm petridishes per batch) and pool. 4