Organoid Dissociation Solution

1. Organoid Recovery: It is recommended to remove the upper culture medium and add an appropriate amount of organoid basal medium when the organoid diameter exceeds 200 μm or when the color turns dark. Use a cell scraper or a 1 mL pipette tip to gently detach the contents from the culture well and transfer them into a centrifuge tube.

2. Organoid Dissociation: Add 5 - 10 times the volume of organoid dissociation solution (relative to the volume of the organoid matrix gel mixture) to the recovered organoids. Gently mix and incubate at 37°C for 1 - 8 min. For single-layer organoids, the dissociation time is typically 1.5 - 3 min, while for multi-layer or larger organoids, the dissociation time is 3 - 8 min.

Note: The dissociation process should be carefully monitored to avoid over-dissociation. Microscopic inspection can be performed during dissociation. If a large number of single cells or cell clusters with a diameter of less than 50 μm are observed, dissociation should be terminated.

3. Termination of Dissociation: Once dissociation is complete, stop the reaction by adding 5 times the volume of organoid basal medium to the organoid suspension.

Note: Fetal bovine serum (FBS) can also be added to ensure cell viability, with a final concentration of 2 - 5%.

4. Organoid Recovery: Centrifuge at 150 - 300 g for 3 min to collect the organoid pellet, discarding the supernatant. Resuspend the organoid pellet in an appropriate amount of organoid basal medium, then centrifuge again at 150-300 g for 3 min. Discard the supernatant and collect the organoids for subsequent culture or freezing.