Organoid Recovery Solution

Pre-cooling the Organoid Recovery Solution on ice before starting the experiment

1. Place the culture dish on ice and carefully aspirate the organoid culture medium, ensuring not to disturb the Matrigel containing the organoids.

2. Add 10 times the volume of pre-cooled organoid recovery solution to each well. For instance, if the Matrigel volume is 5 µL, add 50 µL of recovery solution.

3. Incubate the culture plate at 4°C or on ice for 15 min.

4. Gently scrape the Matrigel using a cell scraper or pipette, transferring the contents of each well to an appropriate tube (use a 1.5 mL EP tube for a single well or a 15 mL centrifuge tube for mult-wells) and incubate on ice for 35 min.

Note: Handle monolayer organoids carefully to avoid cell detachment, which may lead to low recovery rates.

5. Centrifuge at 4°C at a low speed of 25 - 50 g for 3 min. Discard the supernatant and collect the organoid pellet.

Notes: a. Alternatively, incubate the centrifuge tube on ice for 5 - 10 min to let the organoids to settle naturally at the bottom.

b. If a refrigerated centrifuge is unavailable, centrifugation at room temperature is also acceptable, as the organoid morphology will remain stable.

6. Wash the organoids once with 10 times the volume of basal culture medium at room temperature, centrifuge at 4°C at a low speed of 25 - 50 g for 3 min, discard the supernatant, and collect the organoid pellet. Repeat the washing step once.

7. The recovered organoids are ready for subsequent experiments and biochemical analyses.