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)].
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)].
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