Intestinal Crypt Organoid Culture

Organoids were prepared using published protocols 42 (link). Briefly, fragments of small intestine were transferred into crypt isolation buffer (2mM EDTA in PBS) under gentle agitation at 4° for 30 min. The released crypts were resuspended in Matrigel™ (BD) (300-500 crypts/50ml) and cultured in Advanced DMEM/F12 + GlutaMAX™ (Life Technologies) supplemented with 10mM Hepes (Sigma-Aldrich), 1% PEST (Sigma-Aldrich), 1% N2 supplement (Life Technologies), 2% B27 supplement (Life Technologies), murine recombinant epidermal growth factor (50ng/ml), murine recombinant Noggin (100ng/ml) (PeproTech EC Ltd., London, UK) and human recombinant R-spondin1 (1μg/ml) (R&D). Well-differentiated crypt organoids were split into 30 organoids/50μl Matrigel™/well and used for subsequent experiments.

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Huang S.W., Walker C., Pennock J., Else K., Muller W., Daniels M.J., Pellegrini C., Brough D., Lopez-Castejon G, & Cruickshank S.M. (2016). P2X7 receptor-dependent tuning of gut epithelial responses to infection. Immunology and Cell Biology, 95(2), 178-188.

Publication 2016

Matrigel Advanced DMEM/F12 + GlutaMAX™ Hepes N2 supplement B27 supplement human recombinant R-spondin1

Buffer Crypt Edta Epidermal growth factor Hepes Human Isolation Matrigel Murine Noggin Organoids Pest Small intestine Supplement

Corresponding Organization : University of Manchester

Other organizations : University of Pisa

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Variable analysis

independent variables

Fragments of small intestine
Crypt isolation buffer (2mM EDTA in PBS)
Matrigel™ (BD)
Advanced DMEM/F12 + GlutaMAX™ (Life Technologies) supplemented with 10mM Hepes (Sigma-Aldrich), 1% PEST (Sigma-Aldrich), 1% N2 supplement (Life Technologies), 2% B27 supplement (Life Technologies), murine recombinant epidermal growth factor (50ng/ml), murine recombinant Noggin (100ng/ml) (PeproTech EC Ltd., London, UK) and human recombinant R-spondin1 (1μg/ml) (R&D)

dependent variables

Well-differentiated crypt organoids

control variables

Gentle agitation at 4° for 30 min
300-500 crypts/50ml Matrigel™
30 organoids/50μl Matrigel™/well

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