Last part of your question, how many hours you have coated with collagen I? I coated overnight my inserts in another experiment, dried them, sterilized them under UV light in hood, rinse once in PBS just before adding cells, you can rinse with medium once instead of PBS that is not that important.

Subhash C. Juneja Thanks for your suggestion. It turned out to be my isolation was having a problem, hence the cells were not attaching. However, I have fixed it and the coating was working just fine with 1 hour in 37oC incubator and wash with PBS 2 times.

But I am still wondering if AML12 media can be good for primary Hepatocyte, in my experience, the cells were looking just fine in more than 72h

Richard Premont Thank you very much for your suggestion. You were right, resuspending the pellet gently (I did just inverting the tube) helped a lot compared to using a pipette. I have succeeded in isolating the cell since I got quite good attachment and high number of living cells as they form full morphology after 12 hours of isolation. But as you recommended, I will try keeping them in just DMEM high glucose as they gradually lose their characteristics over time and they can not be considered similar to the immortalized one.

I am also worried about culturing them in a long time period but as I am studying about their lipid metabolism I am curious if there is anyways that can allow me to see their lipid droplet accumulation without keeping them for a long time (at least 72 hours)?