Hi Agnieszka, Unfortunately primary hepatocytes do not proliferate invitro therefore passage is not possible. I do not know whether life technologies have some restrictions about passage number. However, We isolate primary hepatocytes ourself and use them fresh without any passage.

Thank you Atta very much. I realize that isolating fresh hepatocytes is better idea, but I have no experience with this method. Hence I decided simply to buy cells for one experiment (Life Technologies: 0 passage).

I use a culture medium consisting of MEM/M199 (3 :1) plus 1 g/L BSA, 2.2 g/L NaHCO3, 5 mg/L insulin, 133 IU/L penicillin, 0.1 mg/L streptomycin, 133 IU/L kanamycin and 10% fetal bovine serum (FBS). In the first 4 h after seeding, the medium also include 50 μM prednisolone 21-hemisuccinate. Hepatocytes are seeded at a density of 105 cells/cm2 in 12-well plastic dishes and incubated at 37 °C under 5% CO2 atmosphere. The medium must be changed every 24 h, and they are OK for at least 96 h. (J Gene Med 2006; 8: 306–313.)

If they are already attached you must change the growth media every day (maybe you can buy the growth media to Life Technologies) and you can work with them for a few days.

There is a Primary Human Hepatocytes webinar coming up that may able to help address questions.

Title: Current State and Future Prospects for Primary Human Hepatocytes in Basic Research Applications Presenter: Edward L. LeCluyse, Ph.D. Principal Scientist, LifeNet Health

Date: Tuesday, June 5, 2018 Times: 1PM (EST) / 12 PM (CST)

Duration: 60 minutes To register for the event, visit: SigmaAldrich.com/PHHWebinar

What you will learn:

• Current challenges with the use of primary hepatocytes for basic research applications
• The state of the art in tissue procurement and hepatocyte isolation techniques; Improved measures for the characterization and validation of primary hepatocytes lots for specific research applications
• Efforts towards creating universal standards and quality metrics for the use of human tissues and cells for basic research applications

Great Kat, thank you for sharing.

Hi Atta,

Can you tell me please for how long your hepatocytes survive in your cultures ? Do they de-differentiate ?

Thank you,

Orly

Check this link maybe useful

Article Long-Term Culture and Coculture of Primary Rat and Human Hepatocytes

regards