] as an additional model, we next generated monolayers from rectal organoids of the twelve donors used in the previous experiments ( Fig. Abbreviations: AUC, area-under-the-curve CCH, carbachol Fsk, forskolin HIS, histamine IBMX, 3-isobutyl-1-methylxanthine ICM, intestinal current measurements Isc, short-circuit currents wt, wild type. Each bar represents two donors, unless stated otherwise by addition of a or b to the corresponding number of CFTR mutation. Averages ± SD of three independent experiments. d) Forskolin-induced and carbachol-induced swelling of (non-)differentiated rectal organoids. c) Representative images of undifferentiated organoids stimulated with 0.8 μM forskolin. Data of three out of twelve donors could not be included due to a different protocol (E60X/4015delATTT (1a) and two S1251N/F508del (3)). Each bar represents two donors except for 1b, genotypes are depicted in d. b) Total changes in Isc (μA/cm 2) after rectal biopsy stimulation with Fsk, IBMX, CCH and HIS. a) Representative tracing of ICM from a wild type rectal biopsy. Chemicals were supplied by Sigma Aldrich unless stated otherwise.ĬFTR function measurements in rectal biopsies (intestinal current measurements, ICM) and rectal biopsy-derived organoids (organoid swelling) of twelve donors, two donors per CFTR genotype/mutation class. Order of compound addition was as indicated in the figures. 3, M + S, Cystic Fibrosis Foundation Therapeutics), dimethylsulfoxide (DMSO) (M + S) and carbachol (S). 3, M + S), CFTRinh-172 (concentrations indicated in Fig. The following compounds were sequentially added to the apical (mucosal, M) and/or basolateral (serosal, S) side: amiloride (S), forskolin (concentrations indicated in Fig. After 30 min of calibration, open circuit potential (PD) and transepithelial resistance (Rt) were measured and equivalent short-circuit (Ieq) currents were calculated every 30 s. Organoid growth medium was replaced by 160 μl and 940 μl of adjusted Ringer's buffer (in mM: 120 NaCl, 20 Hepes, 1.2 CaCl 2, 1.2 MgCl 2, 0.8 KH 2PO 4, 0.8 K 2HPO 4, 5 glucose, pH 7.4) supplemented with indomethacin (10 μM, Sigma) at the apical and basolateral sides, respectively. The MTECC system (24-well format, EP-devices) was used to perform electrophysiological measurements in monolayers cultured under (non-)differentiation conditions. Electrophysiological measurements were either performed directly or growth medium was replaced with organoid differentiation medium for another five days, until the resistances of the monolayers exceeded 800 Ω After four days the measured resistance values exceeded 100 Ω Medium (without Y-27632) was refreshed every 1–2 days. 250,000 cells were seeded per insert, with addition of 100 μl and 600 μl organoid growth medium supplemented with Y-27632 (10 μM, Selleck Chemicals), at the apical and basolateral sides respectively. Seven-day old organoid cultures were trypsinized (TrypLE, Thermo Fisher) for 2 × 2 min at 37 ☌ and mechanically disrupted after each incubation period. 24-Well Transwell HTS plates (3378, Corning) were coated with PureCol (Advanced Biomatrix) diluted 1:100 in Dulbecco's Phosphate-Buffered Saline (PBS) supplemented with calcium and magnesium (Gibco) and incubated for 2 h at 37 ☌/5% CO 2 after which the PureCol solution was removed. Abbreviations: A ( amiloride), AUC ( area-under-the-curve), cAMP ( cyclic AMP), CCH ( carbachol), CF ( cystic fibrosis), CFTR ( cystic fibrosis transmembrane conductance regulator), D ( differentiation culture conditions), DMSO ( dimethylsulfoxide), ENaC ( epithelial sodium channel), FIS ( forskolin-induced swelling), Fsk ( forskolin), GAPDH ( glyceraldehyde 3-phosphate dehydrogenase), HBE ( human bronchial epithelial cells), HIS ( histamine), IBMX ( 3-isobutyl-1-methylxanthine), ICM ( intestinal current measurements), Ieq ( equivalent short-circuit current), Isc ( short-circuit current), LGR5 ( Leucine-rich repeat-containing G-protein coupled receptor 5), MTECC ( Multi Transepithelial Current Clamp), N ( non-differentiation culture conditions), NKCC1 ( Na-K-Cl co-transporter 1), ns ( not significant), PAS ( periodic acid-Schiff), Rt ( transepithelial resistance), RT-q ( reverse transcription quantitative), wt ( wild type) Keywords
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