Organoid growth medium

Primary colonic crypts were isolated from descending aspects of the colons of REF and HBP subjects with gentle cell dissociation reagents (STEMCELL Technologies) including 2 mM EDTA for 90 min. They were grown and maintained as 3D-spheroid cultures in Matrigel (BD Biosciences) containing organoid growth medium (STEMCELL Technologies) with recombinant human Noggin [PeproTech] and EGF [BioLegend], recombinant human IGF-1, FGF-basic [FGF-2; BioLegend] and R-spondin1 [R&D], Y-27632 [STEMCELL Technologies], and A83-01 [Tocris], as described previously [17 (link),20 (link),55 (link)].
Colonic organoids were cultured for 8 days, then treated with 3.0 mM butyrate (Sigma-Aldrich, St. Louis, MO, USA) for 24 h in the following experiments. Selection of the butyrate dose was based on our published data [17 (link)], demonstrating that a 3.0 mM dose was optimal for gene expression without affecting organoid growth [20 (link)]. This is consistent with doses of butyrate in colonic epithelial cells (2 mM) in regulation of the assembly of tight junctions [56 (link)], in Caco-2 cells (10 mM) for apoptosis [57 (link)] and its levels in the gut, portal, hepatic and venous blood [58 (link)].

Biopsies were washed three times with cold PBS and incubated with 2 mM EDTA in PBS for 30 min at 4 °C on a rotating wheel. The supernatant was restored with fresh, cold PBS, centrifuged (200×g, 5 min, 4 °C), and resuspended in Matrigel (Corning, New York, USA). Then 250 µl medium (Organoid Growth Medium, Stem cell, Vancouver, Canada) was added and organoids were cultured at 37 °C in a 5% CO₂ atmosphere. The medium was changed every 2–3 days and organoids were passaged once a week. To obtain organoid monolayers for the PLA assay, the organoid medium was removed, and replaced with Cell recovery solution (Corning, New York, USA), and the plate was put on ice for 20 min to dissolve the Matrigel. The organoids were collected with cold PBS in a 15 mL tube and centrifugated at 200×g for 5 min at 4 °C. The pellet was washed with 5 ml cold PBS and again centrifugated, resuspended in 500 µl TripLE Express (Thermo Scientific, Waltham, USA), and incubated at 37 °C for 15 min. Organoids were dissociated into single cells by pipetting vigorously. The single cells were centrifuged at 200×g for 5 min, 4 °C. The pellet was resuspended in organoid medium, and cells were seeded into precoated (Matrigel, PBS 1:50; 1 h; 37 °C) chamber slides (Thermo Scientific, Waltham, USA).