Culturing Wnt3a/R-spondin/Noggin Organoids

Mouse l-cells that expressed Wnt3a, R-spondin, and Noggin were commercially purchased (ATCC CRL-3276, Genova, Italy), and a conditioned medium (L-WRN) was prepared according to the instructions and protocol of the manufacturer. Culture medium with supplements (CM-S) was prepared using 50% conditioned L-WRN medium and 50% fresh primary culture media: Advanced DMEM/F-12 (cat.12634-010, Invitrogen, Milan, Italy) 1 mM N-Acetyl-l-cysteine (cat. A7250, Sigma, Milan, Italy), 1 × B-27^® supplements (cat.12587-010 Gibco, Milan, Italy), 50 ng/mL Epidermal Growth Factor (cat. PMG8041 Gibco Milan, Italy), 10 mM nicotinamide (cat. N0636, Sigma, Milan, Italy), 10 nM Leu15-gastrin I (cat. G9145 Sigma, Milan, Italy), 500 nM A8301 (inhibitor for ALK4/5/7, cat. 70024-90-7 Sigma, Milan, Italy), 10 µM SB202190 (p38 MAP kinase inhibitor, cat. S7076 Sigma, Milan, Italy), and 10 µM Y-27632 (p160 ROCK inhibitor; cat.1254 Tocris, Milan, Italy) in accordance with the protocols [45 (link),46 (link),47 (link)]. The organoids were cultured with a 300 µL culture medium, which was changed every second or third day.