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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



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...




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