Cryopreserving hepatic organoids can be a key point in your experimental workflow as it offers a pausing point before further differentiation, or to preserve samples for future experiments.

This protocol is for cryopreserving established hepatic organoids in dome cultures grown using the HepatiCult™ Organoid Kit (Human).

Materials

• Healthy hepatic organoid cultures expanded in HepatiCult™ Organoid Growth Medium (Human)
• CryoStor® CS10 (Catalog #07930)
• Advanced DMEM/F-12 (Thermo Fisher 12634028)
• 25% bovine serum albumin (BSA) solution in water
• Sterile 2 mL cryogenic vials
• Sterile 15 mL conical tubes (Catalog #38009)
• Sterile 6-well tissue culture-treated plate (Catalog #38015)
• Sterile 200 µL and 1000 µL pipette tips
• Styrofoam box with ice
• Biohazard safety cabinet
• Controlled-rate cell freezing container (e.g. CoolCell®, Mr. Frosty™ etc.)
• Incubator at 5% CO₂ and 37°C

Preparation

1. Prepare 50 mL of serum-supplemented Advanced DMEM/F-12 (AdvDMEM + BSA):
   • 2 mL of 25% BSA
   • 48 mL of Advanced DMEM/F-12
   • Mix thoroughly and store on ice. Use cold.

   Note: This volume is sufficient to cryopreserve one full 24-well plate. If not used immediately, store AdvDMEM + BSA at 2 - 8°C for up to 4 weeks.

2. Place CryoStor® CS10 and labeled 2 mL cryogenic vials on ice in the biosafety cabinet.

Protocol

1. Check that the Matrigel® domes containing the organoids being cryopreserved are intact. If the dome is intact, proceed to step 2. If the dome is loose, add cold AdvDMEM + BSA to top-up the total volume in the well to 1mL and let sit for 1 minute, then proceed to step 4.
2. Without touching the Matrigel® dome, aspirate and discard the medium in each well being processed.
3. Using a 1 mL pipettor, forcefully add 1 mL cold AdvDMEM + BSA to the center of each Matrigel® dome and let sit for 1 minute.
4. Set the 1 mL pipettor to 1000 µL and vigorously pipette the volume up and down 45 times, taking care not to generate bubbles. This results in mechanical breakdown of hepatic organoids and Matrigel® into smaller fragments of 30 - 100 µm.

   Note: Check the sizes of the fragments generated in the well using a light microscope before proceeding to step 5. If most fragments are larger than 100 µm, triturate until they are 30 - 100 µm.

   Note: If organoid density is low, or if cryopreserving fragments from multiple wells with the same sample or treatment, the contents of multiple wells can be pooled into a 15 mL conical tube at this stage.

5. Vortex the plate containing the organoid fragments in individual wells or the tube containing the pooled organoid fragments gently at medium speed for 5 seconds. To quantify the organoid fragment density in these volumes, immediately transfer 3 x 10 µL from the individual wells or the tube into an empty 6-well plate, creating three separate droplets.
6. Using a light microscope, quantify the number of hepatic organoid fragments in each 10 µL droplet, only counting fragments that are 30 - 100 µm. It is recommended to cryopreserve 1000 - 3000 fragments in each cryogenic vial.

   Note: If the fragment density is too high to count, dilute the fragment suspension with AdvDMEM + BSA and repeat the count.

7. Transfer the desired number of fragments to new 15 mL conical tubes containing 1 mL Advanced DMEM/F-12. Prepare a separate conical tube for each cryogenic vial that will be frozen.

   Example:

   • Three 10 µL fragment counts: 35, 40, 42
   • Average count per 10 µL: 39
   • Volume to transfer 1000 fragments to subsequent passage: 256 µL
   • Desired number of cryovials containing 1000 fragments: 4
   • Prepare 4 x 15 mL conical tubes, each containing 1 mL Advanced DMEM/F-12. Add 256 µL of fragment suspension to each of the four tubes.
8. Centrifuge the tubes at 290 x g for 5 minutes.
9. Without disturbing the pellets, carefully aspirate and discard as much of the supernatant as possible, leaving 5 - 10 µL in the tube. The pellet is often not visible.
10. Place tubes containing pellets on ice. Working with one tube at a time, add 1 mL CryoStor® CS10 and pipette up and down 5 - 8 times to resuspend. Transfer the entire volume into one labeled and cooled cryogenic vial.
11. Working quickly, repeat step 10 for all remaining tubes. Transfer all prepared cryogenic vials into a controlled-rate cell freezing container.
12. Place cell freezing container at -80ºC for 24 - 48 hours, then transfer cryogenic vials to liquid nitrogen storage.

Note: It is recommended to thaw at least one cryogenic vial within 1 - 2 weeks of cryopreservation to verify viability and density of cryopreserved hepatic organoid fragments.