① Organoids cultured for 2-5 days after passaging are used. |
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② Remove medium from the wells. |
Please refer to the video in Method 2《Human intestinal organoid passaging(enzymatic dispersion)》operation①~③ |
③ Add 250 µL of TrypLE™ Express Enzyme (Thermo Fisher Scientific) to the wells and break up the gel with the tip of pipette. |
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④ Collect the entire volume of ③ in a 15 mL low-adsorption tube. |
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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). |
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⑥ Add 9 mL of Basal medium. |
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⑦ Centrifuge (400 g × 3 min., room temperature). |
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⑧ Remove the supernatant (after confirming that the cells have become a pellet). |
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⑨ Centrifuge at 400 g for 3 minutes at room temperature. |
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⑩ Remove the remaining medium with a pipette (confirm that no cells are sucked). |
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⑪ Add 500 µL of cell culture freezing medium to the suspension. |
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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. |
1. Establishment of human intestinal organoids
2. Human intestinal organoid passaging(enzymatic dispersion)
3. Frozen stock production of human intestinal organoids
4. Resuscitation of frozen human intestine organoid