Primary Hepatocyte Culture Protocol

All cell culture procedures must be conducted in a bio-safety cabinet.

Any and all media, supplements, and reagents must be sterilized by filtration through a 0.2 µm filter.

Use aseptic technique to prevent microbial contamination.

Medium

Review the information provided on the Cell Biologics website about appropriate culture media (e.g. serum and other supplements). Use pre-warmed (37°C) cell culture media (30-50 ML, Catalog No. M1365) to seed cells and when changing media.

Coating of Cell Culture Plates or Dishes

Coat sterile culture dishes or flasks with Gelatin Coating Solution (Catalog No. 6950, Cell Biologics) for 0.5 min, then aspirate the excess solution.

Handling of Arriving Live Cells

When you receive the hepatocytes in the culture plates

• Gently aspirate half of supernatant from each well.
• Place dishes in a humidified, 5%-CO₂ incubator at 37°C for 30 min.
• Aspirate supernatant and add 3 ml of fresh medium of M1365, Cell Biologics for each well of 6-wells.
• Place dishes in a humidified, 5%-CO₂ incubator at 37°C for 8-12 hours (overnight).
• Next day, wash cells, remove non-adherent cells with PBS, and add the fresh culture medium until experiments.
• Cells should be checked daily under a microscope to verify appropriate cell morphology.

Cell Recovery from Cryovial

• Quickly thaw cells in cryo-vial by incubating them in a 37°C water bath for <1 min until there is just a small bit of ice left in the vial.
• Promptly remove the vial and wipe it down with 70% ethanol.
• Transfer cells (3 million cells) from the vial to a sterile tube and add 7 ml of pre-warmed Cell Biologics’ Cell Culture Medium (Catalog number M1365, Cell Biologics)
• Carefully pour cell suspension into 2 wells of 6-well-plate (0.7-1.0 million cells per well of 6-well plate)
• Check on the cells after seeding the cells for 4-8 hours.
• After cells attached to the culture dishes about 70-80% confluence (and/or 50-80% cells attach to a plate), gently aspirate supernatant (remove the non-adherent cells) and add 3 ml of fresh medium of M1365 for each well.
• Change culture medium next day and remove non-adherent cells.
• Cells should be checked daily under a microscope to verify appropriate cell morphology.

Note: The numbers of seeding cells in each well may need to be modified according to user’s experience.

Please send us the cell images (>80-90% confluence) if you have any question or problem with cultured cells.