Peter van Mourik

43 Potentiator synergy in rectal organoids in sliders (aperture 0.011 or 0.018 cm 2 ) adapted to micro-Ussing chambers (P2400; Physiological Instruments, San Diego, U.S.A.). After equilibration and repetitive prewashing of biopsies, the following compounds were added in a standardized order to the mucosal (M) or serosal (S) side of the tissue: amiloride (100 μM, M) to inhibit amiloride sensitive electrogenic Na + absorption; forskolin (10 μM; M+S) to activate CFTR-mediated anion secretion; VX-770 (20 μM, but in some F508del / S1251N biopsies 20 – 40 μM; M+S) to potentiate CFTR; genistein (50 μM, but in 1 F508del / S1251N subject 10 and 100 μM; M+S) to further potentiate CFTR; and carbachol (100 μM; S) to initiate the cholinergic Ca 2+ - and protein kinase C-linked Cl - secretion. Crude short circuit current values (μA) were converted to μA cm -2 on the basis of the surface area of the aperture. The average response of the biopsies per subject to each addition was used to calculate the group averages +/-SD (Fig.1b,c). An unpaired T-test was used to calculate statistical differences (Fig. 1b). Crypt isolation and organoid culture from rectal suction biopsies Methods for crypt isolation and human organoid culturing were slightly adapted from protocols described previously 36 . In short, rectal biopsies were washed with PBS and incubated with 10 mM EDTA for 90 - 120 min at 4 °C. Supernatant was harvested and EDTA was washed away. Crypts were isolated by centrifugation and embedded in 50% matrigel (growth factor reduced, phenol-free, BD bioscience) and seeded (~ 10 - 30 crypts in 3 x 10 μl matrigel droplets per well) in 24-well plates. The matrigel was polymerized for 10 - 30 min at 37 °C and immersed in complete culture medium: advanced DMEM / F12 supplemented with penicillin/streptomycin, 10 mM HEPES, Glutamax, N2, B27 (all from Invitrogen), 1 μM N-acetylcysteine (Sigma) and growth factors: 50 ng ml -1 mEGF, 50% Wnt3a-conditioned medium (WCM) and 10% Noggin-conditioned medium (NCM), 20% Rspo1-conditioned medium (RCM), 10 μM Nicotinamide (Sigma), 500 nM A83-01 (Tocris) and 10 μM SB202190 (Sigma). Growth medium was further supplemented with Primocin (1:500; Invivogen). Vancomycin and gentamycin (both from Sigma) were added during the first week of culture.The medium was refreshed every 2–3 days and organoids were passaged 1:4–1:6 every 7–10 days. The forskolin-induced swelling assay Methods to measure forskolin-induced organoid swelling described previously 29 were slightly adapted. In short, rectal CF organoids (passage 1–30) from a 7–10-day old culture were seeded in a flat-bottom 96-well culture plate (Nunc) in 5 μl 50% matrigel commonly containing 20–80 organoids immersed in 100 μl complete culture medium. One day after seeding, organoids were incubated for 30 min with 3 μM calcein-green (Invitrogen) in complete culture medium. After calcein-green staining, 3