This study was approved by the Ethics Committee of Nanjing First Hospital. The patient signed informed consent form prior to surgery. Fresh colorectal cancer tissues were collected after surgical resection and maintained in ice-cold DMEM/F-12 medium with 15 mM HEPES (Gibco) within 2 hours. Tissues were cut into 1-3 mm 3 pieces, washed ten times using pre-chilled PBS, and digested with Gentle Cell Dissociation Reagent (GCDR) (Stemcell Technologies, cat no.07174) for 50 min at 37°C with shaking. After centrifugation at 300×g for 5 min, the supernatant was discarded. The tissues were resuspended using DMEM/F-12 medium with 15 mM HEPES and 2% BSA (Solarbio Life Science, cat no.9048-46-8) and ltered through 70-μm strainers. The number of intestinal crypts were counted. Next, Matrigel Matrix Growth Factor Reduced (GFR), Red-free (Corning, cat no.356231) was diluted with DMEM/F-12 complete medium (as indicated above) at a 1:1 ratio and the crypts were resuspended. Matrigel containing intestinal crypts was dropped onto the center of a pre-warmed 24-well plate without medium (50 μl/well).
The plate was placed in an incubator at 37℃ for 10 min. Subsequently, human organoid medium IntestiCult OGM (Stemcell Technologies, cat no. A8010) was added and was changed every 2-3 days. Passaging was performed 8 days after culturing.
The plate was placed in an incubator at 37℃ for 10 min. Subsequently, human organoid medium IntestiCult OGM (Stemcell Technologies, cat no. A8010) was added and was changed every 2-3 days. Passaging was performed 8 days after culturing.