Colon Organoid Stem Cell Enrichment

Each line was grown as described previously [24 (link),25 (link),26 (link),27 (link),28 (link)] using a split ratio of 1:3 for routine passaging. Organoids were split five days prior to stem cell enrichment and nine days prior to harvesting. Colon organoids were grown in complex medium for a period of five days, replenishing with fresh medium on day three. On day five, medium was aspirated, and 500 µL/well CV medium (10 mL complex medium, 10 µL 5 mM CHIR99021 (Stemcell Technologies: 72054, Vancouver, Canada), and 100 µL 100 mM valproic acid (Stemcell Technologies: 72292)) was added to each well, as previously described [12 (link)]. Following this, organoids were grown for four additional days.