IntestiCult™ Organoid Growth Medium (Mouse)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Optional: co-culture of mouse intestinal epithelial cells and isolated fibroblasts

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Optional: co-culture of mouse intestinal epithelial cells and isolated fibroblasts

Publication protocol

Isolation of mouse intestinal epithelial cells and mesenchymal cells: The intestine was dissected, flushed, opened longitudinally and then cut into 1 cm pieces. The tissues were incubated in HBSS containing 1 mM EDTA, 1 mM DTT, 0.2% FBS, 4–5 times, 10 min each, at 37 °C, 200 rpm. Epithelial cells were released by vigorous shaking, passed through a 70 μm strainer, washed and immediately lysed for RNA isolation. After epithelial cell removal, the remaining stromal part of the intestine was lysed for RNA isolation. For Drop-seq analysis or for FACS-sorting of mesenchymal cells the tissues were processed as above and then incubated in DMEM 10% FBS containing Collagenase XI (300 units/ml, Sigma, C7657), Dispase II (0.1 mg/ml, Sigma, D4693) and DNase II Type V (50 units/ml, Sigma, D8764) for 1 h, at 37 °C, 200 rpm. Cells released after vigorous shaking were passed through a 70 μm strainer and washed with 2% sorbitol. Such cell preparations were directly processed by Drop-seq or by flow cytometry as described below.
Mouse intestinal organoid culture and fibroblast/crypt organotypic co-culture: Crypts were isolated from the last three fourths of the small intestine. The intestine was flushed, cut longitudinally and the villi were scraped off with a glass coverslip. The tissue was then cut into 0.5 cm pieces which were incubated in PBS containing 5 mM EDTA, 0.2% FBS for 30 min at 4 °C on a rocker. Crypts were released by vigorous shaking and were passed through a 70 μm strainer. Six fractions were obtained after vigorous shaking and the ones enriched for crypts were further processed. Crypts were washed by centrifugation at 200g, 100g and 50g and then used for organoid development in domes made by Matrigel (Corning, 356231) and IntestiCult Organoid Growth Medium (Stem Cell Technologies, 06005) according to manufacturer’s guidelines. When indicated, dmPGE2 (Cayman, 14750) dissolved in ethanol was added daily at a final concentration of 0.1 μM. Ethanol was used as a vehicle control for the untreated organoids. Crypts isolated from Yap1ΔIEC mice were cultured in IntestiCult Organoid Growth Medium (Stem Cell Technologies, 06005) supplemented with 0.5 μg ml−1 recombinant mouse epiregulin (RnD 1068-EP-050) or co-cultured with intestinal fibroblasts in OGM without epiregulin. For the assessment of stem cell activity, organoids or spheroids were dissociated into single cells by incubation at 37 °C in 0.25% trypsin-EDTA solution (Gibco, 25200056) diluted 1:1 with DMEM without serum. Numbers of live cells were counted after staining with trypan blue. In each experiment, the same number of live single cells per condition (n = 3,000–11,000) were cultured in domes made by Matrigel (Corning, 356231) and OGM. Intestinal organoids were stimulated with dmPGE2 at a final concentration of 0.1 μM. Ethanol was used as a vehicle control. The ONO-AE3–208 Ptger4 (EP4) inhibitor (Cayman, 14522) dissolved in DMSO was added at a final concentration of 10 μM 1 h before stimulation. Verteporfin (Cayman, 17334) dissolved in DMSO was added at a final concentration of 1 μM 1 h before stimulation. DMSO was used as a vehicle control for ONO-AE3–208 and Verteporfin. Fibroblasts were isolated from the small intestine of mice. The intestine was dissected, flushed, opened longitudinally and then cut into 1-cm pieces. The tissues were incubated in HBSS containing 1 mM EDTA, 1 mM DTT and 0.2% FBS 4–5 times for 10 min each, at 37 °C, 200 rpm. Epithelial cells were released by vigorous shaking. Then, the tissues were incubated in DMEM 10% FBS containing Collagenase XI (300 U ml−1, Sigma, C7657), Dispase II (0.1 mg ml−1, Sigma, D4693) and DNase II Type V (50 U ml−1, Sigma, D8764) for 1 h, at 37 °C, 200 rpm. Cells released after vigorous shaking were passed through a 70-μm strainer, washed and cultured in DMEM with 10% FBS. For co-culture experiments 2 × 104 fibroblasts were seeded in 48-well plates overnight. Freshly isolated crypts (n = 500) were suspended in 1:1 Matrigel (Corning, 356231) and OGM and added as an overlay on the fibroblasts. Crypts and fibroblasts were co-cultured with OGM. When indicated, the ONO-AE3–208 Ptger4 (EP4) inhibitor dissolved in DMSO was added to the co-cultures every second day at a final concentration of 10 μM. DMSO was used as a vehicle control for the untreated co-cultures.


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