Several papers describe isolation of human hepatocytes from steatotic livers.

It may help in finding informations for your problems with fat-containing cells.

http://www.ncbi.nlm.nih.gov/pubmed/21479238/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813921/

Good luck

Some more suggestions:

We worked a few years ago with fish liver hepathocytes and used the followings to prepare fish liver harvesting medium:

Leibovitz L-15 medium (Contains BSA) additioned with 20% FBS, 10% DMSO, 20% Polyvinylpyrolidone and even Hydroxymethyl starch. The main thing, if you don't have an automated harvesting device, is to cool cells in ice containing water at 4°C, then at -20°C moving (returning) tubes from time to time until small ice cristals appear, and then put tubes at -80°C overnight before storing in liquid N2.

May it help?...

Hi Sven,

I had the same problem working with cod. We solved it by starving the fish for a while so they absorb some of the fat. However, this can change the metabolism of the cells. The main problem is that the cells get caught between all the fat drops and can't be centrifuged down. Maybe there is something like a detergent (that doesn't harm the cells of course) that could be added to the isolation medium?

Good luck!

Post collagenase filtration and centrifugation at low temperature should remove most of the fat (floating)?

I can prepare isolated eel (Anguilla japonica) hepatocytes. Fortunately, the liver contains  not so large amounts of lipid. I suggest that after collagenase digestion and filtration the filtrate is then centrifuged over three times. Every centrifugation its supernatant is removed carefully by gentle aspirating. Usually three time centrifugations is enough to obtain clear hepato-parenchymal cells but in your case more centrifugations may be necessary. Furthermore, when you filtrate the suspension after collagenase digestion, it may be better to cool the suspension before filtration.

Hallo Sven,

Have you tryed to centrifuge longer than 1-2 min after isolation or to use density gradient centrifuging? Namely, fat makes hepatocytes light and sedimentation needs  longer time. You can decrease a bit temperature during centriguging but be careful that you keep it in physiological range for the fish you use.

Good luck Sven!

Best wishes,

Maija Pesonen

I can prepare isolated eel (Anguilla japonica) hepatocytes. Fortunately, the liver contains not so large amounts of lipid. I suggest that after collagenase digestion and filtration its filtrate is then centrifuged over three times and the supernatant is carefully removed by gentle aspirating. Usually three time centrifugations are enough to obtain clear hepato-parenchymal cells but in your case more centrifugations may be necessary. Furthermore, it may be better that the suspension after collagenase digestion cool before filtration.