Human Intestinal Organoid Culture Process

① Lyse human intestinal epithelial cells in a 37°C water bath.

② Add 10 mL of Basal medium in a 15 mL tube and add ①.

③ Centrifuge (400 g × 3 min. at room temperature).

④ Remove the supernatant of the culture medium (after confirming that cells have become pellets).

⑤ Centrifuge at 400 g for 3 minutes at room temperature.

⑥ Remove the remaining medium with a pipette (please be careful that no cells are being removed).

⑦ Add 1 mL of Basal medium to suspend the cells.

⑧ Collect a portion of the cell suspension, mix it with an equal volume of Trypan Blue Solution to stain the cells, and count the number of viable cells.

⑨ To seed 10,000-60,000 cells per well of a 24-well plate, collect necessary cells from ⑦ into a 1.5 mL tube and place the tube on ice.

⑩ Add Matrigel^® to the cell suspension from ⑨ to prepare a cell density of 200,000-1,200,000 cells/mL (10,000-60,000 cells/50 µL).
(If Matrigel^® is diluted below 70%, centrifuge the cell suspension in ⑦ and start the procedure again from ⑥.)

※Matrigel^® is a registered trademark of CORNING Incorporated.

⑪ Make a dome by seeding 50 µL in each well of 24-well plate.

⑫ Warm up in a 37°C incubator for 10-15 minutes to gelatinize.

⑬ Add 500 µL/well of Organoid Growth Medium and incubate in an incubator (add slowly along the wall so that the Matrigel^® does not collapses).

⑭ Change medium with Organoid Growth Medium every 2-3 days.