Notes

1.Aspirate media from wells containing the organoids that need to be cryopreserved. Organoids from up to 3 wells can be pooled and processed simultaneously.
Tip: Large organoids with extensive budding will recover best from cryopreservation. Typically, mouse small intestinal organoids in culture for 7-10 days work best. Approximately 250-500 organoids should be cryopreserved per vial or 1 well of a 6-well plate.
2.Add 2 mL of cold Non-enzymatic Cell Dissociation Solution per well.
3.Incubate the plate for 1 minute at room temperature.
4.Use a cell scraper to release the organoids from the plate.
5.Pre-rinse a P1000 tip with Non-enzymatic Cell Dissociation Solution and then break up the domes by pipetting up and down 10 times. Note: Domes and organoids should be broken into small fragments. Do not leave the organoids in Non-enzymatic Cell Dissociation Solution for extended periods or pipet excessively.
6.Transfer the fragmented domes to a 15 mL conical tube.
7.Add a sufficient volume of cold 1% BSA in DMEM:F12 to obtain a total volume of 13 mL.
8.Centrifuge the conical tube at 300 x g for 5 minutes at 4°C.
9.Carefully aspirate and discard the supernatant; do not disturb the pellet.
10.Re-suspend the pellet in undiluted Stem Cell Freezing Medium at approximately 1 mL per well of a 6-well plate.
11.Transfer 1 mL of solution to each cryovial.
12.Place the cryovials in a CoolCell® LX Alcohol-free Cryopreservation Container and transfer the container to a -80°C freezer for 8-24h.
13.Remove the cryovials from the CoolCell® LX Alcohol-free Cryopreservation Container; transfer the cryovials to the vapor phase of liquid nitrogen for long-term storage.