Isolation of human hepatocytes by collagenase perfusion

To perfuse mouse liver and obtain single-cell suspension, the inferior vena cava was cannulated, and the solutions were pumped with an osmotic mini pump (Gilson Minipuls 3) in the following order: 25 ml of Hanks’ balanced salt solution (HBSS) (−/−) (catalog no. H9394; Sigma-Aldrich), 25 ml of HBSS (−/−) supplemented with 0.5 mM EDTA, 25 ml of HBSS (−/−), and 25 ml of HBSS (−/−) supplemented with 5 mM CaCl₂, 0.05% (w/v) collagenase IV (Sigma-Aldrich), and 0.01% (w/v) deoxyribonuclease I (Sigma-Aldrich).

After perfusion, the liver was carefully removed and placed in a Petri dish containing 25 ml of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The blunt end of a scalpel blade was used to break the liver capsule to release the cells into the medium. After collection, the cells were spun down at 50g for 3 min at 4°C. The pellet was resuspended in 21 ml of DMEM and passed through a 100-μm nylon cell strainer. Isotonic Percoll (9 ml) [1 part of 10 × phosphate-buffered saline (PBS) (−/−) with nine parts of Percoll; GE Healthcare] was added to the cell suspension to separate live and dead cells. Live cells were pelleted at 650g for 10 min at 4°C, and the pellet was resuspended in FACS buffer [PBS (−/−) with 5% FBS and 5 mM EDTA]. To delineate between mouse liver cells and human hepatocytes, cells were labeled with phycoerythrin-conjugated anti–human-HLA-ABC (clone W6/32, Invitrogen 12-9983-42; 1:20), biotin-conjugated anti–mouse-H2Kb (clone AF6-88.5, BD Pharmigen 553568; 1:100), and allophycocyanin-conjugated streptavidin (eBioscience 17-4317-82; 1:500). GFP-positive–labeled samples were sorted to a minimal 95% purity using a BD Influx cell sorter. Flow cytometry was performed in the Flow Cytometry Facility, Westmead Institute for Medical Research, Westmead, NSW, Australia. The data were analyzed using FlowJo 7.6.1 (FlowJo LLC).