Hi Charles, ere the technique we are using in our lab:

Primary mouse hepatocytes were isolated by the two-step liver perfusion method. Briefly, mice were anesthetized with Isoflurane and portal vein was punctured. Inferior vena cava was cut through to allow for efficient perfusion and the liver was washed with Liver Perfusion medium preheated to 37°C (Gibco 17701, Invitrogen) for 8–10 min. Then, liver was digested via perfusion with pre-warmed 67 µ/mg Worthington type I collagenase-containing DMEM medium (CellSystems Biotechnologie Vertrieb GmbH) for 5 min. Extirpated livers were transferred into plates filled with pre-cooled DMEM including 10% fetal calf serum (FCS) (4°C). Cell suspension was collected, filtered through 100 µm-cell strainers (BD Falcon 9322799) and centrifuged at 50g for 1 min at room temperature (RT). To increase the viability of isolated primary hepatocyte, the percoll centrifugation method was used. The cells were re-suspended in Percoll/DMEM/PBS (1:1:0.3) mixture and centrifuged at 50g for 15 minutes at RT. Cell viability was examined by the Trypan blue exclusion test. The pellet was washed three times, and the isolated cells were cultured in hepatocyte culture medium (Gibco Hepatozym-SFM 17705) supplemented with 10% FCS, 1% penicillin-streptomycin and L-Glutamine and were kept on 60-mm collagen-coated culture dishes (BD Falcon Biocoat collagen 1 354401) at a density of 4 × 105 cells/4 ml for further studies. The medium was replaced after 4 hours and after overnight culture.

I think you have to use Collagenase to isolate hepatocytes from fresh rat livers

You need to do perfusion with collagenase. Since you need collagen coated culture plates for primary hepatocytes, washing away the collegenase after isolation is crucial.

collagenase. I also have been using Liver Perfusion Medium. Good results.

Hi Charles, ere the technique we are using in our lab:

Primary mouse hepatocytes were isolated by the two-step liver perfusion method. Briefly, mice were anesthetized with Isoflurane and portal vein was punctured. Inferior vena cava was cut through to allow for efficient perfusion and the liver was washed with Liver Perfusion medium preheated to 37°C (Gibco 17701, Invitrogen) for 8–10 min. Then, liver was digested via perfusion with pre-warmed 67 µ/mg Worthington type I collagenase-containing DMEM medium (CellSystems Biotechnologie Vertrieb GmbH) for 5 min. Extirpated livers were transferred into plates filled with pre-cooled DMEM including 10% fetal calf serum (FCS) (4°C). Cell suspension was collected, filtered through 100 µm-cell strainers (BD Falcon 9322799) and centrifuged at 50g for 1 min at room temperature (RT). To increase the viability of isolated primary hepatocyte, the percoll centrifugation method was used. The cells were re-suspended in Percoll/DMEM/PBS (1:1:0.3) mixture and centrifuged at 50g for 15 minutes at RT. Cell viability was examined by the Trypan blue exclusion test. The pellet was washed three times, and the isolated cells were cultured in hepatocyte culture medium (Gibco Hepatozym-SFM 17705) supplemented with 10% FCS, 1% penicillin-streptomycin and L-Glutamine and were kept on 60-mm collagen-coated culture dishes (BD Falcon Biocoat collagen 1 354401) at a density of 4 × 105 cells/4 ml for further studies. The medium was replaced after 4 hours and after overnight culture.

Hi Charles

I agree with all answers that collagenase is the enzyme of choice to isolate rat liver hepatocytes because the liver cells in the hepatic lobules has a framework of reticular fibers (immature collagen fibers) and hepatic lobules are separated by collagenous fibers. The best method to start primary culture is the perfusion method as described by Ozlem.

Good luck

Trypsin works, but only for fetal or neonatal livers

I have always used the collagenase to realize primary cultures of hepatocytes.

In particular the enzymatic dissociation I did with the perfusion method.

The collagenase was of the type 1 and 220mg 50mg hyaluronidase V, dissolved in 30 ml ca2+ free buffer.

I would also recoment collagenase. Be aware of that collagenases could have different ingretiens and that could result in differences in test results.

Hepatocytes are very delicate, I do not know if they like trypsin. I recommend to check articles that describe te technique.

Agree with the above!!! Primary rat hepatocytes do NOT like trypsin. 2-step collagenase perfusion (originally described by Seglen) is the method of choice. Primary hepatocytes are fragile and can easily get damaged or lysed. Also you should not try trypsinizing them in culture plates.

I tried to isolate mouse hepatocytes with trypsine, which seemed to work, at least the liberated cells were looking good, but afterwards they poorly attached to the petridish. I assume that this is due to digestion of surface proteins by trypsin. One of our neighbouring groups liberates hepatocytes without enzyme, just in calcium free perfusion medium.

I agree with Dr. Nobili. Enzymatic dissociation is the better method.

I agree with the above: the enzymatic dissociation is the most effective of the other. But' [lease, be careful : enzymes may be dangerous for the delicate cultures. Use inhibitors and calibrate the enzyme/inhibitor doses in your experiment. Good luck!

We also use the two step collagenase method, but we use Primaria plates instead of collagen coated plates. These plates work as good as coated ones, but save you the trouble of coating and plates are always ready to use immediately.

I would try to use TrypLe Select which is a recombinant trypsin of high purity. It is less toxic to mammalian cells than any other native enzymes used for tissue digestion. Life technology does not recommend use of inhibitor for this enzyme. I have not seen any publication mentioned this enzyme preparation for hepatocyte isolation, maybe others have?