Hepatocyte Use & Handling

Handling of Fresh Hepatocytes:

Fresh hepatocytes should be used upon receipt. The cells are shipped on wet ice – do not allow them to warm up or come to room temperature before use. If removed from the shipping container, place them into a 4°C refrigerator until use. Refer to the Fresh Hepatocyte use and plating protocol.

Fresh Hepatocyte Use & Plating:

Prior to removing the cells from the shipping container or refrigerator prepare the hood as follows

  • Have all media completed and ready to use.
  • Have the Trypan Blue solution prepared for the appropriate dilution (1:5)
  • Have the cell culture vessel readily available for seeding.
  1. The cell suspension should remain cold until you are ready to use it!
  2. During shipment the hepatocytes will settle out of suspension. Resuspend the cells by gently inverting the shipping vial repeatedly until the cells have uniformly resuspended. Transfer them to an appropriate centrifuge tube.
  3. Centrifuge the tube at 60G for 4 minutes.
  4. Gently aspirate all of the supernatant being careful not to disturb the cell pellet.
  5. Add 5-10 mls of warm media and re-suspend the pellet using a gentle rocking motion. Add the media by pouring along the side of the tube, not directly onto the cell pellet.
  6. Determine the cell count using the Trypan Blue Exclusion method. A 1:2 or 1:5 dilution is recommended.
  7. Adjust cellular density to the desired concentration for your application using room-temp seeding media.
    • For plating mouse hepatocytes, an initial seeding density of 0.40 × 106 cells/ml is recommended.*
    • For plating rat hepatocytes, an initial seeding density of 0.70 × 106 cells/ml is recommended.* * You may need to visually assess the seeding density for optimal results. IMPORTANT -- Over-seeding rodent hepatocytes can lead to cell death!

      Representative Seeding Densities. Mouse, Rat

  8. Once the proper seeding concentration has been established, add the cell suspension to your plate (collagen-coated tissue culture plates are recommended). The following volumes are recommended for each format:
    • 6 well: 2mL/well
    • 12 well: 1mL/well
    • 24 well: 0.5mL/well
    • 48 well: 0.250mL/well
    • 96 well: 0.125mL/well
  9. Place the plate into a tissue culture incubator (37°C, 5% CO2). Hold the plate flat on the incubator shelf and gently shake it in a right to left and then forward to back pattern (↔, ↑↓) or figure 8 motion to evenly disperse the cells throughout the plating surface. Avoid circular motions that will result in piling up the cells.
  10. Allow 2-3 hours for attachment.

    Hepatocytes after attachment.
    Hepatocytes after attachment.

    Once the cells have sufficiently attached carefully aspirate the seeding media and replace with warm (37°C) maintenance media. IMPORTANT -- Pipette the new media into the well along the side-wall of the well – do NOT pipette the media directly onto the newly formed monolayer!
  11. If your application requires a sandwich culture:
    • Mouse hepatocytes - Aspirate the maintenance media and replace with cold media (containing Matrigel®) at the first media change (2-3 hours after plating)
    • Rat hepatocytes – Aspirate the maintenance media and replace with cold media (containing Matrigel®) at the first media change (2-3 hours after plating)

      Note: Overlaying Rat hepatocytes after 2-3 hours can result in the formation of chords (as shown in the picture). If a more confluent monolayer is desired, then replace the seeding media with warm maintenance media after 2-3 hours and then overlay ~8-10 hours later!

      hepatocytes

    • Replace media every 24 hours thereafter with warm (37°C) maintenance media.