Human Intestinal Organoid Culture Process

① Remove medium from wells at 7-10 days of culture in ① ＜Method 1. Establishment of human intestinal organoids＞.

② Add 500 µL of TrypLE™ Express Enzyme (Thermo Fisher Scientific) to the wells and collapse the gel with the tip of the pipette.

※TrypLE™ is a registered trademark of Thermo Fisher Scientific.

③ Collect the total volume of ② in a 15 mL low-binding tube.

④ Treat 15 mL tubes in a warm water bath at 37°C for 5-10 minutes and disperse cells by using the pipette (about 20-30 times).

⑤ The dispersion of the organoids is checked under a microscope to determine if the reaction time should be extended or further dispersed with the pipette.

⑥ Add 9 mL of Basal medium.

⑦ Centrifuge (400 g × 3 min. at room temperature).

⑧ Remove the supernatant of the culture medium (after confirming that the cells have become pellets).

⑨ Centrifuge (400 g × 3 min. at room temperature).

⑩ Remove the remaining medium with a pipette (should be careful that no cells are being removed).

⑪ Add 300-500 µL of Basal medium to suspend the cells.

⑫ 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.

⑬ To seed 1,000 cells per well of a 48-well plate, collect necessary cells from ⑪ into a 1.5 mL tube and place the tube on ice.

⑭ Add Matrigel^® to the cell suspension from ⑬ to prepare a cell density of 50,000 cells/mL (1,000 cells/20 µL).
(If Matrigel^® is diluted below 70%, centrifuge the cell suspension in ⑪ and start the procedure again from ⑩.)

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

⑮ Make a dome by seeding 20 µL in each 48-well plate.

⑯ Warm up in a 37°C incubator for 10-15 minutes to gelatinize.

⑰ Add 250 µL/well of Organoid Growth Medium and incubate in an incubator (add slowly along the wall so that the Matrigel^® does not collapses).

⑱ Change medium with Organoid Growth Medium every 2-3 days.

※For uniform seeding of cells
After operation ⑤, set a cell strainer (mesh size: 20 µm) on a 15 mL tube and filter the cell suspension with it. Then, start with operation ⑥.