Intesticult ogm mouse basal medium

Enteroid cultures were generated as described previously [30 (link)]. Murine jejunal tissues were harvested by dissection and any fat tissue was removed. Tissues were sliced longitudinally to expose luminal contents, washed three times in phosphate buffered saline (PBS), and cut into ~0.5 cm sections. Sections were incubated in ethylenediamine tetraacetic acid (EDTA) (30 mM in PBS) (Corning, Loughborough, UK) for 5-min cycles and subsequent vigorous shaking for 15 s in PBS dissociated crypt units. Fractions from each cycle were assessed by microscopy and those containing intact crypts largely free from contaminating villi were passed through a 70-μm strainer to remove villi. Isolated crypts were counted, centrifuged at 300×g for 5 min at 4°C and re-suspended at 10 crypts/μl in 70% Growth Factor Reduced, Phenol Red Free, Matrigel^® (Corning) and 30% IntestiCult OGM Mouse Basal Medium (StemCell, Cambridge, UK) and plated. The Matrigel^® cultures were polymerized at 37°C in 5% v/v atmospheric CO₂ for 20 min, and overlaid with IntestiCult OGM Mouse Basal Medium. Enteroid medium was replaced every 3–4 days and cultures were split on days 7 or 8 at a ratio of 1:3 or 1:4.

Small intestines were opened longitudinally, cut into 0.5-cm pieces, and washed with ice-cold PBS. Intestinal crypts were isolated by treatment with Gentle Cell Dissociation Reagent (StemCell Technologies, Cambridge, MA) and then passed through a 70-μm cell strainer. Equal numbers of isolated crypts were mixed 1:1 (vol/vol) with Matrigel (BD Biosciences, Franklin Lakes, NJ) and plated in 48-well plates. Matrigel was polymerized by incubating it at 37°C for 10 min, and 400 μl of Intesticult™ OGM Mouse Basal Medium (StemCell Technologies, Cambridge, MA) was added into each well. The culture medium was replaced every 2–3 days and the organoids were passaged by mechanical dissociation every 7 days. To determine the effect of L. acidophilus, crypts were cultured in the presence of heat-killed L. acidophilus, the formation of organoids was observed at passage 2 using a light microscope and the presence of functional cells was analyzed by immunofluorescence followed by confocal microscopy. Intestinal organoids were harvested using Cell Recovery Solution (Corning, NY). The organoid pellets were resuspended in TRIZOL reagent (Ambion, CA) for RNA isolation.

The small intestine of 4-week-old C57BL/6 wild-type mice was cut into small pieces and washed several times with cold PBS. The pieces were then incubated with Gentle Cell Dissociation Reagent (Stem Cell, Vancouver, Canada) for 15 min at 20 °C, and crypts were detached from basal membrane by vigorous shaking. After incubation, crypts enriched in the supernatant were passed through a 70 μm strainer and centrifuged at 300× g for 5 min at 4 °C. The cells were counted, and then, about 200 crypts in each well were resuspended by 25 μL Matrigel (Corning, NY, USA) and 25 μL IntestiCult^TM OGM Mouse Basal Medium (Stem cell, Vancouver, Canada), and then plated in 24-well plates. After polymerizing at 37 °C for 20 min, the culture medium supplemented with penicillin–streptomycin (100 U/mL) was added into the gel in each well. The medium was changed every 2–3 days.

The small intestine was dissected from male C57BL/6N mice (ORIENT Bio, Korea) or LGR-EGFP-IRES-CreERT2 (LGR5-GFP) mice, which were kindly provided by Professor Mi-Na Kweon (College of Medicine/Asan Medical Center, Korea). Intestinal crypts were isolated using the Gentle Cell Dissociation Reagent (StemCell Technologies, MA). The tissue was incubated with 0.1% bovine serum albumin (BSA), and the cell suspension was passed through a 70-μm cell strainer. The isolated crypts were observed under a microscope (CKX53, OLYMPUS, Japan). The crypts were mixed with Matrigel (BD Biosciences, NJ) and Intesticult^TM OGM Mouse Basal Medium (StemCell Technologies) at a ratio of 1:1, and 20 μl of the suspended crypts was plated in 48-well plates. After polymerization by incubating at 37 °C for 20 min, 400 μl of Intesticult^TM OGM Mouse Basal Medium was added, and the plate was placed in a humidified incubator (5% CO₂) at 37 °C. The culture medium was replaced every 2 days, and the organoids were passaged every 5 to 6 days.
For passaging, the culture medium was removed, and the organoids were recovered from the Matrigel using the Cell Recovery Solution (Corning, NY). After mechanical disruption by pipetting, the suspended crypts were transferred to microtubes and centrifuged at 850×g for 5 min. The crypt pellets were mixed with Matrigel and Intesticult^TM OGM Mouse Basal Medium and cultured.