Cryopreserving hepatic tissue samples adds flexibility to your experimental design and workflow. Cryopreserved tissue can be stored long-term in liquid nitrogen providing flexible starting points for your experiments, and allowing you to limit the number of passages your samples experience.

This protocol is for cryopreserving non-tumorigenic hepatic tissue for organoid culture using controlled rate freezing containers.

Materials

• Liver tissue sample
• DMEM/F-12 + 15 mM HEPES (Catalog #36254)
• Fetal bovine serum (FBS)
• Sterile Petri dishes
• Sterile 50 mL conical tubes (Catalog #38010)
• Sterile 2 mL cryogenic vials
• Dissection tools
• Styrofoam box with ice
• CryoStor® CS10 (Catalog #07930)
• Controlled-rate cell freezing container (e.g. Corning® CoolCell® (Catalog #200-0642), Mr. Frosty™ etc.)

Preparation

1. Prepare 30 mL of Wash Solution:
   • 0.3 mL FBS
   • 29.7 mL DMEM/F-12 + 15 mM HEPES
   • Mix thoroughly and store at 2 - 8°C for up to 4 weeks. Use cold.
2. For cryopreservation in freezing medium, place CryoStor® CS10 and labeled 2mL cryogenic vials on ice in the biosafety cabinet.

Protocol

1. Under aseptic conditions, transfer the liver tissue sample to the Petri dish containing 20 - 25 mL of cold Wash Solution.
2. With the tissue sample submerged in Wash Solution, use scissors to cut into small (~3 - 5 mm) pieces.
3. Using sterile forceps, transfer 1 - 2 tissue pieces that are 3 - 5 mm in size into each cryogenic vial.
4. Add 1 mL freezing medium (e.g. CryoStor® CS10) to each cryogenic vial.
5. Transfer vial(s) to a controlled rate cell freezing container. Place the freezing container at -80ºC for 24 - 48 hours.
6. Transfer cryogenic vials to liquid nitrogen storage until ready for further processing. Do not refreeze tissue once thawed.