① Dissolve the cell stock in a 37°C warm water bath. |
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② Add 10 mL of Basal medium in a 15 mL tube and add ① (use a low-adsorption tip and low-adsorption tube due to easy adsorption). |
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③ Centrifuge (400 g × 3 min. at room temperature). |
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④ Remove the supernatant of the culture medium (after confirming that the cells have become pellets). |
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⑤ Centrifuge (400 g × 3 min. at room temperature). |
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⑥ Remove the remaining medium with a pipette (should be careful that no cells are being removed). |
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⑦ Add 30-50 µL of Basal medium to suspend the cells. |
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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. |
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⑨ To seed 1,000 cells per well of a 48-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 50,000 cells/mL (1,000 cells/20 µL). |
Please refer to the video in 《Method 1 Establishment of human intestinal organoids》operation⑩ |
⑪ Make a dome by seeding 20 µL in each well of 48-well plate. |
Please refer to the video in 《Method 1 Establishment of human intestinal organoids》operation ⑪ | ⑫ Warm up in a 37°C incubator for 10-15 minutes to gelatinize. |
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⑬ 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). |
Please refer to the video in 《Method 1 Establishment of human intestinal organoids》operation ⑬ |
⑭ Change medium with Organoid Growth Medium every 2-3 days. |
Please refer to the video in 《Method 1 Establishment of human intestinal organoids》operation⑭ |
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