Hepatocyte Use & Handling

Thawing/Plating Instructions for Cryopreserved Hepatocytes:

Prior to beginning the thawing/plating process, prepare the hood as follows:

  • Have all media completed and ready to use: ~75mls of room-temp media per cryo-vial.
  • Have the Trypan Blue solution prepared for the appropriate dilution (1:5)
  • Have the cell culture vessel readily available for seeding.
  1. Remove the vial from the shipping container or freezer and immediately transfer to a pre-warmed, 37° C waterbath. Transferring the vial from shipper to waterbath should take no more than 10 seconds.
  2. Place the vial into the water, but do not completely submerge the vial, be careful to keep the cap above the water (See illustration). Gently shake the vial back and forth to achieve even thawing while continuously monitoring the contents. Remove the vial when the contents have thawed to the point that only a small ice crystal remains. This process should take no more than 2 minutes.

    Cryopreserved Hepatocytes: Cryopreserved cells

  3. Once the cell suspension has thawed, wipe off the outside of the vial with 70% ethanol. Pour the cell suspension into a 50ml conical tube containing ~40mls of media (seeding media). Rinse the vial with 1 ml of media to recover any remaining cells and add to the 50ml conical.
  4. Centrifuge the tube at 50G for 3 minutes.
  5. Gently aspirate all of the supernatant being careful not to disturb the cell pellet.
  6. Add 5mls of room-temp 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.
  7. Determine the cell count using the Trypan Blue Exclusion method. A 1:2 or 1:5 dilution is recommended.
  8. 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!

    Cryopreserved Hepatocytes: Representative Seeding Densities

  9. 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
  10. 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.
  11. Allow 4-5 hours for attachment.

    Cryopreserved Hepatocytes: 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!
  12. If your application requires a sandwich culture:
    • Mouse hepatocytes — Aspirate the maintenance media and replace with cold media (containing Matrigel®) 3-4 hours after the first media change
      (8-9 hours after initial seeding).
    • Rat hepatocytes — Aspirate the maintenance media and replace with cold media (containing Matrigel®) 6-8 hours after the first media change
      (11- 13 hours after initial seeding).
    • Replace media every 24 hours thereafter with warm (37°C) maintenance media.