Cryopreserving Human Liver Tissue for Organoid Culture
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. CoolCell®, Mr. Frosty™ etc.)
Preparation
- 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.
- For cryopreservation in freezing medium, place CryoStor® CS10 and labeled 2mL cryogenic vials on ice in the biosafety cabinet.
Protocol
Note: For best results, tissue should be stored at 4°C in an organ transfer solution (e.g. Advanced DMEM/F-12, HypoThermosol) and processed within 48 hours of isolation.
- Under aseptic conditions, transfer the liver tissue sample to the Petri dish containing 20 - 25 mL of cold Wash Solution.
- With the tissue sample submerged in Wash Solution, use scissors to cut into small (~3 - 5 mm) pieces.
- Using sterile forceps, transfer 1 - 2 tissue pieces that are 3 - 5 mm in size into each cryogenic vial.
- Add 1 mL freezing medium (e.g. CryoStor® CS10) to each cryogenic vial.
- Transfer vial(s) to a controlled rate cell freezing container. Place the freezing container at -80ºC for 24 - 48 hours.
- Transfer cryogenic vials to liquid nitrogen storage until ready for further processing. Do not refreeze tissue once thawed.
