Calcein green

To quantify CFTR function in the genetically corrected intestinal organoids, we conducted the FIS-assay. This was performed in duplicates at three independent culture time points (n = 3) according to previously published protocols (Boj et al, 2017 (link); Vonk et al, 2020 (link)). In brief, intestinal organoids were seeded in 96-well culture plates in 4 μl of 50% Matrigel. Each Matrigel dome contained roughly 20–40 organoids and was immersed in expansion medium. The day after, organoids were incubated for 30 min with 3 μM calcein green (Invitrogen) to fluorescently label the organoids and stimulated with 5 μM forskolin. Every 10 minutes, the total calcein green–labeled area per well was monitored by a Zeiss LSM800 confocal microscope, for 60 min while the environment was maintained at 37°C and 5% CO₂. A Zen Image analysis software module (Zeiss) was used to quantify the organoid response (area under the curve measurements of relative size increase in organoids after 60 min forskolin stimulation, t = 0 min baseline of 100%).

Methodology for organoid swelling was adapted from an existing protocol (Boj et al., 2017 ). All experiments were performed on the Zeiss LSM‐880 multiphoton confocal microscope with temperature control (37°C) and humidity chamber. Intrahepatic organoids were plated in 96‐well plates in 3 µl drops (1:2 biliary organoid media to Matrigel) 1–2 days prior to imaging and supplemented with 100 µl of biliary organoid media with 10 μM Y‐27632 dihydrochloride. On the day of the experiment, one vial of calcein green (50 μg; Invitrogen, Thermo Fischer) was thawed and dissolved in 5.1 μl of DMSO. The resuspended calcein green (2.5 μl) was added to 580 μL of organoid media. 10 μL of this final solution was then added to each well for imaging and allowed to incubate for 30 min. Approximately 30 min prior to the experiment, media was aspirated and replaced with Krebs–Ringer solution [118.9 mM NaCl, 25 mM NaHCO₃, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 2.4 mM K₂HPO₄, 0.6 mM KH₂PO₄, and 5 mM dextrose in 5% CO₂ (vol/vol), pH = 7.4]. Baseline measurements were obtained. Forskolin (final concentration =10 μM) or DMSO (final dilution = 1:1000) was added to the wells and organoids were monitored for 1 h with an image acquisition interval of 5 min. Segmentation and area measurement for each well were performed through the Zen software (Zeiss) and reported as a total area per well.

Five days after organoid trypsinization, 1 mg/ml dispase II (Invitrogen) was added to the medium of the organoids and these were incubated for 15 min at 37° C to digest the BME. Subsequently, organoids were mechanically dissociated by pipetting, filtrated using a 40 μm nylon cell strainer (Falcon), resuspended in 75% BME/growth medium (40 organoids/μl) prior plating of two 10 μl drops on Nunc™ Lab-Tek™ II Chamber Slide™ Systems. After plating culture medium containing either 1 μM of afatinib, 1 μM of selumetinib, a combination of 1 μM of afatinib and 1 μM of selumetinib or DMSO was added. The labtek plates were mounted on an inverted confocal laser scanning microscope (Leica SP8X) and imaged using a 10X objective. For visualization of cell viability, organoids were incubated with 16.2 μΜ Hoechst 33342 (Life Technologies) and 1.5 μM DRAQ7™ (Cell Signaling #7406) for 30 min at 37° C prior imaging.
For the GAP domain knock out CRISPR screen, organoids were imaged by an inverted routine microscope (Nikon Eclipse TS100) using a 4X or 10X objective. For calculating organoid count and size, organoids were incubated for 45 minutes with 500 ml culture medium containing 5 μM calcein-green (Invitrogen). For the quantification of the organoid size and count, FIJI analysis software was used.