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

  1. Prepare 30 mL of Wash Solution:
  2. 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.

  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.
  • Document #PR000047
  • Version 1.0.0
  • May 2021