Penicillin

The human epithelial cell lines HT-29 and Caco-2 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and grown as described (23 (link)). Four human primary colonic cell culture from three different donors were performed as described (24 (link)). Briefly, PBS-washed colonic tissues were digested with 0.5 mg/ml of collagenase type XI. The crypts were plated onto Matrigel coated plates and cultured for 24 h in DMEM 24 mM glucose supplemented with 10% FCS, 2 mM L-Glutamine, 50 U/mL penicillin, 50 U/mL streptomycin, and Y-27632 (Tocris). The day after plating, media was rinsed with fresh media and replaced with culture media with or without butyrate 2 mM. Human tissues were obtained from the Human Research Tissue Bank at the Addenbrooke's Hospital, Cambridge under the license 09/H0308/24.

All human studies were approved by local ethical review committee (09/H0308/24). Human tissues were obtained from the Human Research Tissue Bank at Addenbrooke's Hospital, Cambridge, UK, and processed within the same day.
Colonic crypts (from colon and rectum for mice samples and from distal colon or rectum for human) were isolated and cultured as previously described [12] , [13] (link). Briefly, tissue was thoroughly washed with cold PBS, outer muscle layers discarded, and samples minced. Crypts were then isolated by incubation in collagenase type XI (Sigma) at 0.4 mg/mL for mouse tissue and 0.5 mg/mL for human tissue and cultured on Matrigel (BD Bioscience) coated plates in high glucose with 10% FBS, 2 mM Glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 10 μM Y-27632 dihydrochloride (Tocris). Cultures were processed the day after plating after visual inspection for seeded cell density.

For adipogenesis assays, cells were cultured in αMEM containing 15% FBS, 100 U/ml penicillin/streptomycin, 2 mM L-glutamine, 10^–6 M dexamethasone (TOCRIS), 50 μM indomethacin (Sigma), and 100 μg/ml insulin (Invitrogen). The medium was replaced every 2–3 days, and after 21 days, cells were stained with Oil Red O (Sigma). For osteogenesis assays, cells were cultured with fresh osteogenic differentiation media containing 10 mM β-glycerolphosphate (Sigma) and 50 μg ascorbic acid (Sigma). The medium was replaced every 2–3 days, and after 28 days, cells were stained with Alizarin Red S (Sigma) or Von Kossa (Abcam). For chondrogenic assays, 7.1 × 10⁴ cells were resuspended in a 10-μl droplet and plated as a micromass in the center of a 48-well plate. Cells were incubated for 1.5 h at 37 °C in a humidified 5% CO₂ incubator. Chondrogenic differentiation medium containing high-glucose DMEM supplemented with 1% ITS-Premix (Thermo Fisher Scientific), L-ascorbic acid-2-phosphate (Sigma) (0.1 mM), dexamethasone (Sigma) (1 × 10⁷ M), proline (Sigma) (400 mg/ml) and BMP-2 (Sigma) (100 ng/ml) was added onto the micromass droplet. The medium was replaced every 2–3 days, and after 9 days, micromass pellets were collected for Alcian Blue staining.