Intesticulttm organoid growth medium

Adult human normal colonic tissues were obtained through the Vanderbilt Cooperative Human Tissue Network. As described before,^{26 (link)} crypts were collected and suspended in growth factor reduced Matrigel (#356230, Corning Incorporated, Corning, NY) and overlaid with media IntestiCult^TM Organoid Growth Medium (#06010, STEMCELL Technologies, WA, USA). For colonoid subculture, colonoids were broken down by vigorously pipetting and cultured as above. All colonoids became budding at 3–4 days of culture. Colonoid treatment was performed by supplementing with p40F, p40N180, or p40N120 at 100 ng/ml in medium for 48 h before stopping experiments. Colonoids were removed from culture dishes by using cell recovery solution (354253, Corning), and collected organoids were fixed in 10% neutral buffered formalin for 20 min followed by embedding in 2% agarose. Agarose plugs containing organoids were then embedded in paraffin to allow hematoxylin and eosin staining and immunostaining.

Crypts were resuspended in IntestiCult^TM Organoid Growth Medium (STEMCELL Technologies, Vancouver, Canada), plated (approximately 50–100 crypts per 40 μL drop of 50% Matrigel), and overlaid with IntestiCult^TM Organoid Growth Medium. Then, the organoids were maintained in a cell incubator at 37 °C containing 5% CO₂.

For isolation of crypt epithelial cells, EDTA treated intestinal tissue fragments were vigorous suspension by using a 1 ml blunt tip with cold PBS. The supernatant was the villous fraction and discarded (repeat twice). After a further vigorous suspension of tissue sediment, the supernatant was enriched for crypts epithelial cells. This fraction was passed through a 70 μm cell strainer to remove residual villous material. Isolated crypts were centrifuged at 150–200 g for 3 min to separate crypts from single cells. For organoid formation, isolated crypts were cultured using IntestiCultTM Organoid growth Medium (STEMCELL Technologies, Vancouver, BC, Canada), growth factor reduced Matrigel (Corning, Bedford, MA, USA) and DMEM/F12 (Welgene, Gyeongsan, Republic of Korea) according to manufacturer’s instruction.

Antral tumors from gp130^FF mice were used to establish tumor-epithelial organoids. Organoids were established and maintained in IntestiCult^TM Organoid Growth Medium (StemCell Technologies) according to manufacturer's protocols. Established organoids were stimulated with 100 ng/ml IL-11 or PBS control for 4 h and then processed for gene expression analysis via qPCR. For assessment of organoid growth, PBS or IL-11 stimulated organoids were monitored over 4 days.

The colon was flushed gently, opened longitudinally, and then cut into 2 mm pieces. Gentle cell dissociation reagent (Stem cell, Cat# 07174, Canada) was used to isolate colonic crypts by incubating for 20 minutes at room temperature (15–25 °C). Crypt fractions were purified through successive centrifugation steps. Then the same volume of complete IntestiCult^TM organoid growth medium (Stemcell, Cat# 06005, Canada) and undiluted Matrigel (Corning, Cat# 356231) were added to culture intestinal organoid. Details of this model have been described previously by Sato T et al.^{53 (link)}.

Ex vivo organoid culture was established with whole crypts freshly isolated from mouse intestinal tissues (adapted from [33 (link)]). After a 30 min incubation of the minced tissue with 3 mM EDTA at 4 °C, the crypts were pelleted and passed through a 70 µm strainer. Crypts were seeded in Matrigel 1:1 IntestiCult^TM organoid growth medium (StemCell) and maintained in IntestiCult^TM medium for 5 days before passaging. Organoid development was followed day by day with classic optical microscopy and pictures were taken at a 4, 10, and 20× magnification with a ZEISS Primovert microscope in brightfield. Organoids were pelleted at day 5 for RNA analysis.