Polarized Intestinal Organoid Monolayers

The 2D HIO culture method was adopted from a previously published protocol using patient-derived intestinal organoids generated from fresh rectal biopsies (Zomer-van Ommen et al, 2018 (link); Vonk et al, 2020 (link)). Mature 3D HIO were dissociated with trypsin (TrypLE; Thermo Fisher Scientific), diluted with growth medium (IntestiCult Human OGM, STEMCELL Technologies) supplemented with Y-27632, and seeded onto Purecol pre-coated transwell inserts (Costar 3470, 6.5 mm diameter, 0.4 μm pore size; Corning) for generating a polarized monolayer (Zomer-van Ommen et al, 2018 (link)). Y-27632 was removed 1 d post-seeding and the growth media were changed every other day. Ussing chamber studies were conducted 4–5 d post-seeding. Cultures were incubated with CFTR modulators (Table 1) 18–24 h before Ussing experiments.