n/a n/a (Primary hepatocytes (liver cells))

(Cryopreservation of Primary Hepatocytes)


This protocol is adapted from current protocols in toxicology and is for the cryopreservation of primary hepatocytes from any laboratory animal. It is not species-specific and may therefore require optimization for best results.


• Isolated hepatocytes
• Fetal bovine serum (FBS), heat-inactivated
• Dimethyl sulfoxide (DMSO)
• Hepatocyte storage vials (must withstand −196°C)
• Microprocessor-controlled programmable freezing chamber (e.g., model 1010; Forma Scientific) with thermometer
• Liquid nitrogen storage unit


1. Centrifuge isolated hepatocytes 3 (±1) min at 40 to 60 × g, room temperature.
2. Discard supernatant fraction by aspiration with a vacuum aspirator.
3. Add a sufficient volume of FBS to bring concentration to 11 × 106 viable cells/ml and gently resuspend cell pellet.
4. Slowly add DMSO dropwise to a final concentration of 10% (v/v) and mix cell suspension by gentle inversion.
5. Divide the homogenous mixture into aliquots in hepatocyte storage vials.
6. Cryopreserve hepatocytes in a microprocessor-controlled programmable freezing chamber according to the following stepwise freezing program. Use a temperature probe to measure the temperature of a sample containing only FBS and 10% (v/v) DMSO.
    6a. Cool −1°C/min until sample reaches −4°C
    6b. Cool −25°C/min until chamber reaches −25°C (sample temperature should be approximately −8° to −10°C)
    6c. Warm 15°C/min until chamber reaches −12°C
    6d. Cool −1°C/min until chamber reaches −40°C
    6e. Cool −10°C/min until chamber reaches −90°C
    6f. Hold −90°C until sample reaches −90°C.
7. Transfer vials to a liquid nitrogen storage unit.

To thaw:
1. Remove one or more vials of cryopreserved hepatocytes from the liquid nitrogen storage unit.
2. Place in a 37°C shaking water bath until sample is partially thawed and can be poured from the vial.
3. Gently pour thawed hepatocytes into a sterile 50-ml centrifuge tube containing 28.2 ml DMEM+ and 11.8 ml of 90% isotonic Percoll.
4. Add DMEM+ to obtain a final concentration of 21.5% (v/v) Percoll.
5. Centrifuge hepatocytes 5 (±1) min at room temperature at 60 x g.


Mudra, D. R. and Parkinson, A. 2001. Preparation of Hepatocytes. Current Protocols in Toxicology. 8:14.2.1–14.2.13.

Added by: Carlton Hoyt on July 25, 2012

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