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

(Cryopreservation of Primary Hepatocytes)

Intro/Description:

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.

Materials:

• 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

Protocol:

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.

References:

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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