Plating Hepatocytes

Plated cryopreserved human hepatocytes after 72 hours at 10x magnification from donor HUM4033
Plated cryopreserved human hepatocytes after 72 hours at 10x magnification from donor HUM4033


Human hepatocytes can be plated, cultured and analyzed for different drug interactions or other liver function experiments. Follow this protocol to ensure your cells will have optimal yield and function. Please note that these hepatocytes cannot be passaged. 



  1. Coat your plate of choice with PureCol® at least two hours prior to running this protocol by following this protocol (Coating Plates with PureCol®);
  2. Make sure you have properly thawed and counted your hepatocytes by following the Thawing Hepatocytes and the Hemocytometer Cell Counting protocols; 
  3. Remember to spray everything down with 70% ethanol and wipe it with Kimwipes before going into the BSC;
  4. Warm hepatocyte plating medium to 37˚C in the water/bead bath;
  5. The recommended cell density for hepatocyte plating is 1 x 106 cells/mL, so add the appropriate amount of plating medium to ensure you have that final concentration;
    1. Example: If you have counted a total of 5 million cells in your suspension, you will need a total of 5 mL of medium. The formula is: total cells/cell concentration = amount of media to add to cell pellet (in mL). 
  6. Add the appropriate volume of cell stock to each well in your plate (refer to the table below for well plate working volumes);
PlateWell Working Volume
6-well2 mL
12-well1 mL
24-well500 µL
48-well200 µL
96-well100 µL
  1. Place plate in the incubator, gently shaking it in order to ensure cell distribution throughout the plate;
  2. Repeat shaking at 15, 30 and 45 minutes post-seeding;
  3. After 60 minutes, replace plating medium with fresh medium using the indicated working volumes;
  4. Incubate cells for 4-6 hours;
  5. Replace medium with hepatocyte culture medium daily, including different components to your media depending on your experimental goals.

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