Intesticult organoid growth medium ogm

Intestinal organoids were isolated and cultured in vitro using
IntestiCult^™ Organoid Growth Medium (Mouse) following
the manufacturer’s instructions. Briefly, 20 cm of small intestine
proximal to the stomach was harvested and longitudinally opened. The tissue
was cut into 2 mm pieces and washed with ice-cold PBS 15 to 20 times with
gently shaking until the supernatant was clear. The tissue was then digested
with gentle cell dissociation reagent for 15 min at room temperature on a
rocking platform at 20 rpm. Tissue pieces were collected and resuspended in
PBS with 0.1% BSA. Crypts were isolated by gently shaking and then filtered
through a 70-μm strainer. The supernatant containing crypts was
collected and pelleted by centrifugation at 200× g for 3 min.
Approximately 200–500 crypts were embedded in Matrigel (Sigma) per
well of a 24-well plate and submerged in IntestiCult^™Organoid Growth Medium (OGM, Stem Cell Technologies) with included
supplements. Organoids were subcultured every 6–7 days at 37°C
in a 5% CO₂ environment with a 1:4 splitting ratio.

Isolation and culture of ileal organoids were performed as previously reported^{26 (link)}. Briefly, ileum isolated from mice was dissected, and washed with Dulbecco’s PBS 10 times. Then, the ileal fragments were incubated with Gentle Cell Dissociation Reagent (STEMCELL Technologies) to separate the crypts and villi from the intestinal basement membrane. After centrifugation, the crypts were isolated by filtration (70 μm) and resuspended in a 1:1 mixture of Matrigel (Corning) and IntestiCult organoid growth medium (OGM) (STEMCELL Technologies) at a density of 6000 crypts/mL. A droplet of 50 μL containing 300 crypts was placed into the center of each well of a prewarmed 24-well plate, forming a dome. After the domes had solidified, 750 μL of OGM was added to each well. The crypts were cultured at 37 °C under 5% CO₂, and the medium was refreshed every 3 days.
For compound C (CC, AMPK inhibitor) treatment, ileal organoids were incubated with control, nicotine (1 μg/mL) or nicotine plus CC (20 μM) for 12 h. For GW4869 treatment, HFHCD-fed WT mice were treated with nicotine water or nicotine water plus 10 mg/kg GW4869 (by daily gavage) for 2 weeks, and then organoids were isolated and cultured. The ileal organoids were cultured for 7 days and treated with GW4869 (10 μM) and nicotine (1 μg/mL) for the last 3 days before being harvested.

Intestinal biopsies were used to establish adult colonic stem cell-derived epithelial cell cultures (colonoids) that were plated as monolayers on 0.4 μm Transwell^® inserts (VWR, cat#: 76313–906), as previously described [46 (link), 52 (link)]. Briefly, colonoids from Matrigel domes were dislodged using a cell scraper and incubated in Cultrex Organoid Harvesting solution (R&D Systems, cat#: 3700-100-01) at 4°C for 90 minutes. Cells were washed, treated with TrypLE Express (Thermo Fisher Scientific, cat#: 12604021) for 1 minute at 37°C, washed and resuspended in IntestiCult^™ Organoid Growth Medium (OGM; STEMCELL Technologies, cat#: 06010) supplemented with 50 μg/mL gentamicin (Thermo Fisher Scientific) and 10 μM Rho kinase inhibitor Y-27632 (STEMCELL Technologies, cat#: 72304). Transwells^® were pre-treated with 34 μg/mL human collagen IV (Sigma-Aldrich, cat#: C5533) and cells from 1.2 to 1.4 domes were seeded per Transwell^®. Cells were incubated at 37°C (v/v 5% CO₂) and media were changed every 2 days. Growth was monitored by light microscopy for confluence and transepithelial electrical resistance (TEER) was measured with a Millicell^® ERS-2 Volt-Ohm meter, as previously described [46 (link)].