Primary Human Hepatocytes (Pooled and Plateable) thawing may be tricky in the way for your best experimental results. The improper thawing may result in poor pool viability or insufficient cell number, in addition to physiological changes and sterility issues. In order to help you avoid it, we want to publish our standard protocol for hepatocytes thawing, tested through multiple experiments and optimization attempts. Video support is attached for your convenience.

Materials and Equipment:

  • Water bath with heating
  • Serological pipettes
  • Micropipettes
  • Centrifuge with rotor for 50 mL tubes
  • +37 incubator with 5% CO2
  • Laminar Box
  • Sterile Box with ice
  • Liquid Nitrogen tank
  • Counting chamber
  • Collagen 1 coated 24- or 96-well plates

Caution!

It is critical to store obtained cells in the temperature lower than -150 °C.

DO NOT store cells in the dry ice. Use personal protective equipment: lab coat, gloves and goggles when working with cell cultures.

Reagent Preparation

  1. Prepare Hepatocyte Medium: Mix William’s E Medium (450 mL) with 50 mL of Fetal Bovine Serum (final concentration 10%), add Bovine insulin (4 µg/mL) and Antibiotic-Antimycotic solution to final concentration 1%. Note: HepExtend Supplement could be used as serum-free alternative to FBS and Bovine insulin for preparation of Hepatocyte Medium. The Medium should be stored at 4 °C until use.
  2. Dilute 5.04 ml of Percoll with 0.56 ml of 1.5 M NaCl water solution.
  3. Prepare Hepatocyte Thawing Medium by adding Percoll solution to Hepatocyte Medium.
  4. The volume of Thawing Medium for 1 vial of Plateable Hepatocytes is 20 mL: Add 5,6 mL of Percoll solution to 14,4 mL of Hepatocyte Medium and mix well. The Thawing Medium should be preheated to 37 °C before thawing procedure.
General scheme of human hepatocytes thawing

Seeding of Hepatocytes

For seeding and cultivation of cells Hepatocyte Medium should be used.The cells should be seeded on Collagen 1 coated plates. Depending on well area, the number of seeded hepatocytes varies:our convenience.

  1. For 24-well plates seed cells in concentrations of 500 thousand cells/well.
  2. For 96-well plates use 50,000-70,000 cells/well depending on experimental requirements.
video