We are isolating hepatocytes from mouse liver and trying to culture them in 6 or 24 well plates. The isolation is carried out according to the protocol and plating is done using William E medium with 10% FBS and 1% PEST. We use to coat the plates with collagen 1mg/ml. But after 3 hours and more the cells are loosely attached to the plates. We are sure these cells are hepatocytes because we stained them with the appropriate markers. Maybe something is missing in the medium which is essential for attachment?
Hello Giulio Preta
You could prepare a stock of collagen I in 50 mM HCl for a final concentration of 1 mg/ml. Warm in a 37°C water bath until the collagen dissolves completely. Prepare a working stock of 60 μg/ml in sterile water.
Pipet 1ml of the above collagen working solution into each well, replace lid, and place in the 37°C incubator for 4 hr. Then rinse each well three times, each time with 2 ml HBSS to neutralize the acid and aspirate the last rinse.
Dispense 2 ml of complete William’s Medium E into each well and leave in the incubator for 30 min to equilibrate with the matrix. Then you need to add the desired number of cells per well containing complete William’s Medium E. You will have to replace the medium after 4 hr. with 2 ml/well fresh William’s Medium E. This step is an important step for hepatocyte cultures as it will help to remove debris produced during collagenase digestion and cell dissociation. You may then perform your experiments only after an overnight incubation.
In case, you do not succeed with the above protocol, there are many other substrates that may be used for hepatocyte cultures which includes fibronectin, laminin, and combinations of these with collagen.
Hope this information helps!
Best.
I think you should apply a mild heat not higher than 40oC for about
20 minutes, after applying the collagen solution. That should enhance attachment of the cells.
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