① Lyse human intestinal epithelial cells in a 37°C water bath. |
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② Add 10 mL of Basal medium in a 15 mL tube and add ①. |
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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 cells have become pellets). |
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⑤ Centrifuge at 400 g for 3 minutes at room temperature. |
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⑥ Remove the remaining medium with a pipette (please be careful that no cells are being removed). |
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⑦ Add 1 mL 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. |
![]() Images of viable (white) and dead (dark blue) 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). |
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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. |
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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). |
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⑭ Change medium with Organoid Growth Medium every 2-3 days. |
The following figures are microscopic images (2x objective lens, scale bar = 100 µm) at day 6 and day 10 of culture
(seeding density: 30,000 cells/50 µL)
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