① Remove medium from wells at 7-10 days of culture in ① <Method 1. Establishment of human intestinal organoids>. |
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② Add 500 µL of TrypLE™ Express Enzyme (Thermo Fisher Scientific) to the wells and collapse the gel with the tip of the pipette. |
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③ Collect the total volume of ② in a 15 mL low-binding tube. |
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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). |
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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. |
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⑥ Add 9 mL of Basal medium. |
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⑦ Centrifuge (400 g × 3 min. at room temperature). |
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⑧ Remove the supernatant of the culture medium (after confirming that the cells have become pellets). |
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⑨ Centrifuge (400 g × 3 min. at room temperature). |
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⑩ Remove the remaining medium with a pipette (should be careful that no cells are being removed). |
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⑪ Add 300-500 µL of Basal medium to suspend the cells. |
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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. |
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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). |
Please refer to the video in 《Method 1 Establishment of human intestinal organoids》operation ⑩ |
⑮ Make a dome by seeding 20 µL in each 48-well plate. |
Please refer to the video in 《Method 1 Establishment of human intestinal organoids》operation ⑪ |
⑯ Warm up in a 37°C incubator for 10-15 minutes to gelatinize. |
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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). |
Please refer to the video in 《Method 1 Establishment of human intestinal organoids》operation ⑬ |
⑱ Change medium with Organoid Growth Medium every 2-3 days. |
Please refer to the video in 《Method 1 Establishment of human intestinal organoids》operation ⑭ |
※For uniform seeding of cells |
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The following images are microscopic images (2x objective lens, Scale bar, 100 µm) at day 2, day3, day6 and day 10 of passaging.
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