To generate organoid from a single erythropoietin-producing hepatocellular receptor B2, or FEB2-positive intestinal stem cell, carry out fluorescence-activated cell sorting based on the FEB2 expression, and divide the cells obtained into four groups, high, medium, low and negative. After collecting the FEB2 high cell pellet with centrifugation at 390G for three minutes at four degree Celsius, embed the single sorted FEB2 high cells in the ECM by pipetting, then seed the cells on a 24-well plate at 100 singlets per 40 microliters of ECM per well. Incubate the 24-well plate for 15 minutes at 37 degrees Celsius and 5%carbon dioxide for the polymerization of the ECM.
Next, cover the ECM with 500 microliters of culture medium containing 10 micromolar row-associated kinase, or rock inhibitor for the first two days to maintain the FEB2 high cells. Manually inspect the cells using an inverted microscope at 40X magnification and observe viable organoids with spheroid formation and crypt protrusion. Self-organizing crypt villa structures reminiscent of the normal small intestine were recreated from single FEB2 high cells.
By day five of culture, spheroid-like structures formed, and from day seven to day nine, evagination of the spots to form crypts occurred.
