Primary culture hepatocytes of mouse - (Apr/09/2006 )

Does anyone could help me to perform primary mouse hepatocytes cultures?
I performed hepatocytes isolation by perfusing the liver trough the left ventricular with 10 ml of PBS and after with 15 ml of collagenase type I (sigma, 0.05% in PBS), both solution were at 37 C. When the liver was white I washed it in PBS and after I minced it in collagenase for 10 min at 37 C.
By using a 10 ml syringe, I aspirated the cells and the small pieces and I ejected them into the dish. Then, I filtered (50 micron net) the clear medium with the cells with a filter placed on the top of a falcon-50 ml tube (the tube is on ice). I Added some additional medium (3-4 ml William E medium 10% FCS, Glutammine 1mM, Insulin 100 nM) into the Petri dish and I repeated the procedure 2-3 times. When the big pieces can move trough the syringe, I used the needle (Microlance 3, big hole) and I toke all the pieces of the liver and the medium. After the filtration I centrifuged the cells at 700 g for 5 min. Then I aspirated the medium and I replaced it with the same volume of medium, then I centrifuged at 500 g for 5 min. Finally I removed the supernatant, I added new medium and centrifuged another time at 400 g for 5 min. Cellular viability (90%) was tested by trypan blue exclusion, no erythrocytes were found. I suspended the cells in small volume (0.7 ml per 35 mm diameter well, 1 million of cells per well). I used dish or plates coated with collagen type I (sigma).
After 5 hours, the cells attached (40-45% of total cells), but the day after the isolation they are all detached. Why? Do I have to add some other factor in the medium? Is the time of digestion appropriate?
All suggestions are welcomed.
Thank you very much
Matteo Ricchi
University of Lausanne
Switzerland

Hi,

I just made a little experimet by lysing a mouseliver and putting the mixed cells in dishes,
coated and uncoated one....the cells grow best at the UNCOATED wells....maybe you try that...


Greetz

K_R

That's true, we also found the UNCOATED dishes better than coated one.