Hello Shamir Montazid

Try following this protocol.

1. Thaw the vial rapidly in approximately 2 minutes in a 37°C water bath. Some ice crystals should remain in the vial.

2. Transfer the vial contents to a 15 mL conical tube containing 9 mL ADF medium.

3. Rinse the inside of the vial with 1 mL ADF medium and transfer the medium to the same conical tube containing 9 mL of ADF medium.

4. Centrifuge the conical tube at 300 x g at 4°C for 5 minutes.

5. Carefully aspirate the supernatant without disturbing the pellet.

6. Re-suspend the pellet in 100% Matrigel (pipet up and down 10 times).

7. Dispense approximately 8-10 droplets (10-15 µL per droplet) into each well of the pre-warmed 6-well plate. Avoid creating bubbles. The droplets should retain their shape. If the droplets flatten and spread out to coat the well, it is possible that the plate was not warm enough or the droplets were not dispensed properly.

8. Place the plate containing the droplets in a 37°C, 5% CO2 humid incubator for 15 minutes to polymerize the droplets.

9. Add 2 mL of complete growth medium supplemented with 10 µM ROCK inhibitor, Y-27632 to each well for post-thaw recovery. After 24-hour in culture

Y-27632 is no longer required in the culture medium.

10. Perform a complete medium change every 2-3 days.

I hope this will be helpful.

Best.

Hello Shamir,

Your protocol looks fine. From my standpoint, you should also check if there are any mistakes that happened in your freezing step. For example, did you prewarm the Mr. Frost before putting your cryovials in? Or try to freeze more cells in one cryoviail. By the way, we never freeze the organoids just after 2 days of passage.

Hope this will help you.

Best.