Tissue Dissociation Solution

Thaw the Dissociation Solution and Supplement (20×) at 4°C in advance and mix thoroughly. Prepare the tissue dissociation solution in a sterile. Mix thoroughly and keep on ice before use.

Note: a. After the initial thawing, it is recommended to aliquot the supplement (20×) appropriately to avoid repeated freeze-thaw cycles.

b. The prepared tissue dissociation solution should be used as soon as possible. It can be stored at 4°C for up to 24 h. If not used in the short term, it can be stored at -20°C, but it is recommended to use it within one month.

1. Tissue Pretreatment: Use surgical scissors or a scalpel to cut the tissue into 1 - 3 mm³ fragments.

2. Tissue Dissociation: Add an appropriate amount of tissue dissociation solution according to the size of the tissue fragments, typically 20 - 50 times the tissue volume. After mixing thoroughly, incubate in a 37°C incubator for dissociation. As different samples may vary in tissue source individual differences, the required dissociation time may differ. The typical dissociation time ranges from 30 to 120 min.

Note: The dissociation process should be carefully monitored, as excessive dissociation can reduce the efficiency of organoid formation. During dissociation, microscopic examination can be performed. The dissociation can be terminated when a large number of single cells or cell aggregates with a diameter of less than 70 μm are observed.

3. Termination of Dissociation: After confirming that dissociation is complete, add fetal bovine serum (MCE Catalog No. HY-T1000) to the tissue suspension, with a final concentration of 2 - 5%. Gently pipette to mix and stop the dissociation.

4. Cell Collection: Filter the cell suspension through a mesh or centrifuge to collect the cells. Wash the cells 2 - 3 times with organoid basal medium, centrifuge at 200 - 300 g for 3 min, and collect the cells for organoid culture.