Primary Human Hepatocytes Thawing and Seeding: Tips and Tricks

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

Reagents:

● William’s E Medium (Thermo Fisher)

● Fetal Bovine Serum (Sigma)

● Bovine insulin (Sigma)

● HepExtend Supplement 50X (Thermo Fisher)

● Percoll (Cytiva)

● Antibiotic/Antimycotic 100X (Thermo Fisher)

● Trypan Blue Solution (Thermo Fisher)

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.

Pic. 1. General scheme of human hepatocytes thawing

Thawing of Hepatocytes

1) Remove the required number of cryovials with frozen cells from the liquid nitrogen tank.

2) Quickly place the cryovials in a 37°C water bath (do not use an incubator). Slightly open and then close the vial to remove the excessive Liquid Nitrogen.

3) While holding the tip of the bottle, mix gently, not allowing water to penetrate through the cap. DO NOT submerge the cryotube completely in water.

4) Thaw the vial of cells in a water bath until half of the ice pellet remains (this usually takes about 80-120 seconds).

5) Immediately remove the vial from the water bath. Wipe the outside of the bottle with an alcohol wipe. Place the thawed vial in the laminar box.

6) Gently transfer the contents of the cryovial to preheated Hepatocyte Thawing Medium.

7) Carefully rinse the cryotube with 1 ml of Thawing medium and transfer the remaining cell suspension to a vial. Repeat this step 2-3 times.

8) Gently mix the cell suspension in the vial (DO NOT vortex)

9) Centrifuge at 200g/ 6 min/ at room temperature.

10) Carefully aspirate the supernatant without disturbing the cell pellet.

11) Resuspend the cell pellet in 3-4 ml of cold Hepatocyte Medium.

12) DO NOT use small tips to resuspend cells, it’s better to use 5 mL pipette for more gentle resuspending of hepatocyte pellet.

13) Measure the exact volume of the cell suspension with a 5 mL graduated pipette. Cell suspension should be stored on ice or at +4°C during all next procedures.

14) Check cell concentration and viability. For in-process control as well as quality control of the end product, cell count and viability could be determined by light microscopy and trypan blue exclusion.

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:

● For 24-well plates seed cells in concentrations of 500 thousand cells/well.

● For 96-well plates use 50,000-70,000 cells/well depending on experimental requirements.