IntestiCult™ Organoid Growth Medium (Human)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Replace culture medium every 2 days	After removing the culture medium, RNA can be extracted with Trizol. For immunofluorescence, organoids can be fixed with 4% PFA for 30 min, permeabilized, and blocked for non-specific binding with 3% BSA prior to incubation with primary antibodies.

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Replace culture medium every 2 days
Downstream tips
After removing the culture medium, RNA can be extracted with Trizol. For immunofluorescence, organoids can be fixed with 4% PFA for 30 min, permeabilized, and blocked for non-specific binding with 3% BSA prior to incubation with primary antibodies.

Publication protocol

Intestinal organoids were cultured according to the protocol from StemCell Technologies (Cambridge, MA). Small intestine was harvested from p9 pups mice and cut into small segments. Intestinal crypts were isolated by digestion with Gentle Cell Dissociation Reagent (StemCell Technologies, Cambridge, MA) for 15 min and pelleted by centrifugation. Crypts were then re-suspended in Matrigel (Corning, New York) and transferred into 24-well plates. After polymerization, mouse IntestiCult organoid growth medium (StemCell Technologies, Cambridge, MA) supplemented with penicillin-streptomycin (100U/ml) was overlaid on the gel in each well. Organoids were maintained in a 37 °C incubator with the culture medium replaced every 2 days. Organoids were exposed to PBS, or Wnt7b (200 ng/ml in the culture medium) for 7 days. Control organoids were also grown in medium with and without Wnt, according to a previously published organoids culture medium protocol30. Organoids were imaged daily, and their surface area was calculated using Image J software. RNA was extracted with Trizol after removing culture medium. For immunofluorescence, organoids were fixed with 4% PFA for 30 min, permeabilized, and blocked for non-specific binding with 3% BSA prior to incubation with primary antibodies.
Human intestinal organoids were cultured as described above, with human IntestiCult organoid growth medium (StemCell Technologies, Cambridge, MA) and 10 μM Y-27632 (StemCell Technologies, Cambridge, MA), a ROCK inhibitor was added to the medium for primary culture.

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Manufacturer protocol

Download the product protocol from STEMCELL technologies for IntestiCult™ Organoid Growth Medium (Human) below.

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