How do I optimize 384-well cultures of hepatocytes?

The liquid handling challenges associated with low-volume plates, including 384-well and smaller plate formats, must be managed with extra care to prevent excessive loss of iCell® Hepatocytes 2.0 during the culture process.

To minimize cell losses in 384-well 2D culture formats, we recommend you:

  • Plate at a density of 30,000 cells/well. Each 384-well plate has approximately one-third of the surface area of a 96-well plate. Note that up to 300 wells can be plated using the ≥9 million viable cells in one iCell Hepatocytes 2.0 cryovial. Plating at a lower density could result in deterioration of the culture over time.
  • Maintain a medium volume of 40 - 60 μl/well of Plating Medium or Maintenance Medium.
  • Follow the standard medium change schedule for 96-well plates according to our User’s Guide.
  • Avoid touching the bottom of each well during medium changes. Set the liquid handler’s probe height so the probe’s tip will not touch the well bottom; ensure you don’t touch the well bottom with the pipette tip if manually pipetting.
  • Use a low pipetting speed when performing medium changes. Set the liquid handler’s dispensing speed to a low flow rate to prevent detaching cells from the well bottom; touch the pipette tips to the side of the well and dispense slowly down the side of the well if manually pipetting. After dispensing, spin the plate in a centrifuge to bring the medium to the bottom of the well.
  • Perform medium changes by removing all but 20 μl of spent medium and adding fresh medium to make up the volume difference. In an example using 50 μl of medium in each well, remove 30 μl (50 μl minus 20 μl) of spent medium and add 30 μl of fresh medium.
  • Avoid using wells along the outer edges of the plate as they are prone to evaporation issues that cause culture deterioration, especially if culturing for longer than 5 days. We recommend using only the inner 240 wells for culturing cells and filling the outer two rows and columns with medium only.
  • Minimize liquid handling steps whenever possible. Perform addition-only steps without washing to minimize cell disruption, especially for cells subjected to compound treatment.
  • Perform a dilution rather than 100% medium change when a wash is necessary. For example, remove all but 10 - 20 μl of medium, add 50 μl of wash, and repeat 2 - 3 times to dilute.
  • Use tissue culture plates pre-coated with collagen I for cell attachment. Do not manually apply a fresh coating of collagen I.
  • Invert the plate on absorbent paper and centrifuge to decant the medium for endpoint assays where sterility is no longer required.
  • Perform washes with RPMI (without supplements), which is similar to the Plating Medium and Maintenance Medium, instead of PBS, which can exacerbate detachment.


  1. iCell Hepatocytes User's Guide