Protocols


Primary hepatocyte isolation

Isolation of primary murine hepatocytes

Preparation:

  • 1. Animal, 6-8wks male C57BL/6 mice
  • 2. Instruments
    • 1) BIO RAD peristaltic pump, model# EP-1 Econo Pump
    • 2) water bath (37°C)
    • 3) 24GA 0.75IN 0.7x19mm yellow, closed IV catheter (BD 383511)
    • 4) Surgical forceps, scissors
    • 5) 100/70µm cell strainer(BD)

Medium:

  • 1) Perfusion medium (GIBCO # 17704-024)
  • 2) Liver Digest Medium (GIBCO 17703-034), follow manufacture instruction to store the medium.
    • Divided to 35ml/ Mouse, incubate 30-60 min in 37°C before use.
  • 3) Washing(cold)/Plating medium (individual components can be supplemented separately)
    • William’s E Medium (WEM, GIBCO A1217601)
    • Supplemented with GIBCO Cat# CM3000 (enough for 500ml culture/kit)
  • 4) Maintenance medium
    • WEM supplemented with GIBCO # CM 4000 (enough for 500ml culture/kit), but due to possible inhibition of hepcidin production, WEM with 5% serum may be used instead.
  • 5) Percoll medium, 100% (Sigma P1644-500ML, supplemented 1XPBS, actual Percoll-90%).
    • 40% Percoll: (see below, hold at 4° C)-one mouse needs 20-40ml.
    • ------4 parts 100% percoll + 6 parts plating medium (actual Percoll is 36%)

Perfusion procedures

  • 1) Prepare pump, mouse and reagents.
    • Dissolve collagenase for 30-60min before digestion, temperature 37°C (not to go over 42 °C, also not necessary to use higher than 37 °C)
    • Rinse pump with 70% alcohol, sterile water and perfusion buffer.
    • Clean abdominal area fur with 70% alcohol.
    • Make midline incision to expose abdominal site.
    • Place catheter into portal vein.
  • 2) Start perfusion pump at a rate of 4.5 ml/minute with perfusion medium, immediately cut SVC or IVC to let blood out for about 5 minutes.
  • 3) Stop pump, then move tubing to liver digest medium, continue for 6-7 minutes.
  • 4) Remove liver and place in a 100 mm plate filled with cold 20ml washing medium.
  • 5) Tear liver into pieces with tips or forceps.
  • 6) Pour cells through 100/70µm filters into 50ml centrifuge tubes, add additional 20ml cold wash medium to plate and filter into same tube.
  • 7) Centrifuge tubes at 50Xg for 3 minutes at 4°C.
  • 8) Decant the supernatant and add 20ml 40% cold percoll to each tube, mix with pipette.
  • 9) Centrifuge at 150-200Xg for 7 minutes at 4°C. (After this step, viable hepatocytes will be at the bottom of the tubes)
  • 10)Discard supernatant, wash 1-2 times with 20ml wash medium (as step 7)
  • 11) Resuspend washed cells with 10-20 ml plating medium and count cells using hemacytometer.
  • 12) Seed cells at the following concentration in collagen coated plate
    • 2.2X105 cells/ 6-well
    • 1.0X105 cells/ 12-well
  • 13) Place plates in 37°C incubator with 5% CO2 for 2.5-3.5 hours. Wash once to remove dead cells, replace medium with maintenance medium.
  • 14) Change medium next day, start experiment same day or next day, cells can survive for ~ 4 days.