Culture Hepatocytes

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. M1265) 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 10 min, then aspirate the excess solution before seeding cells.

Handling of Arriving Live Cells

When you receive the live cells in the cell culture medium

• Centrifuge cells at 50g for 2 minutes.
• Aspirate the supernatant and resuspend the cell pellet in Cell Culture Medium (Catalog No. M1265, Cell Biologics).
• Seed cells gently in plates or dishes coated with Gelatin Coating Solution (Catalog No. 6950, Cell Biologics). Cell seeding density is as below recommended.
• Place dishes or plates in a humidified, 5%-CO₂ incubator at 37°C for 6-12 hours.
• Wash cells once with PBS and add the fresh culture medium until experiments.
• Cells should be checked under a microscope to verify appropriate cell morphology.

Recommended Fresh-Cell Seeding:

• 3 million cells are seeded 2 wells of a 6-well plate;
• 6 million cells are seeded 4 wells of a 6-well plate;
• 6 million cells are seeded to a 48-well plate;
• 6 million cells are seeded per well of a 96-well plate;

Note: The number of seeding cells in each well may need to be doubled by using frozen vials.

Handling Freshly Plated Hepatocytes

·       Place plate in biosafety cabinet after receiving cells.

·       Aspirate all shipping medium from each well.

·       Replace with the appropriate volume of hepatocyte medium (Catalog No. M1269, Cell Biologics).

·       Place the plates in a 5% CO₂ incubator at 37°C.

·       Allow the hepatocytes to culture overnight (12-18 hours) before using them.

·       Replace the maintenance medium daily.

Note:

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