Human Intestinal Organoid Culture Process

① Organoids cultured for 2-5 days after passaging are used.

② Remove medium from the wells.

③ Add 250 µL of TrypLE™ Express Enzyme (Thermo Fisher Scientific) to the wells and break up the gel with the tip of pipette.

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

④ Collect the entire volume of ③ in a 15 mL low-adsorption tube.

⑤ Put the 15 mL tube in a warm water bath at 37°C for 5-10 minutes, and disperse the cells by pipetting (about 20-30 times).

⑥ Add 9 mL of Basal medium.

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

⑧ Remove the supernatant (after confirming that the cells have become a pellet).

⑨ Centrifuge at 400 g for 3 minutes at room temperature.

⑩ Remove the remaining medium with a pipette (confirm that no cells are sucked).

⑪ Add 500 µL of cell culture freezing medium to the suspension.

⑫ Add the entire amount to the frozen vial and store in a CoolCell (CORNING) at -80°C overnight, and store at -150°C the next day.