Several lymphocyte subpopulations reside in the normal adult human liver. Liver lymphocytes mainly include a large number of T cells, B cells, NK cells and natural killer T (NKT) cells, which are distinct from the peripheral blood lymphocytes (PBL). In these cell populations, NK cells are about 10-15% of total hepatic lymphocytes.

1. Under deep methoxyfluran anesthesia, a needle was carefully inserted into the portal vein. The liver was perfused with 20 ml PBS (pH7.0), and then removed to a petri dish.
2. The liver was thoroughly dissected and gently passed through a 200-gauge stainless steel mesh and then suspended in culture medium containing 100 ml/l fetal calf serum (FCS).
3. The above cell suspension was centrifuged at 1,500 rpm.
4. The pellet was resuspended in 40% Percoll solution containing 100 U/ml heparin, and then loaded on the layer of 70% Percoll solution followed by centrifugation at 2,000 rpm for 20 min at room temperature.
5. The cells were aspirated from the Percoll interface and harvested by centrifugation and washed twice with Hanks’ Balanced Salt Solution (HBSS) containing 50 ml/l FCS.
6. Under deep anesthesia, a needle was inserted into the portal vein.
7. The inferior caval vein was cut to enable blood out-flow.
8. The liver was perfused with 20 ml liver perfusion medium, followed by an injection of 5 ml liver digestion medium mainly containing 0.05% collagenase IV and 0.001% DNase I, then the liver was removed and gently pressed through a mesh.
9. The liver cell suspension was collected, and parenchymal cells were separated from mononuclear cells (MNCs) by centrifugation at 500 g for 5 min.
10. MNC populations were purified by centrifugation through a Percoll gradient.
11. Cell pellet was collected, washed in PBS, and resuspended in 40% Percoll in complete culture medium.
12. The cell suspension was gently overlaid onto 70% Percoll and centrifuged for 20 min at 750 g.
13. MNCs were collected from the interface layer, washed twice in PBS, and resuspended in proper medium for further process.

Reference
Curry MP, Norris S, Golden-Mason L, et al. Isolation of lymphocytes from normal adult human liver suitable for phenotypic and functional characterization. J Immunol Methods.
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Trobonjaca Z, Kr oger A, Stober D, et al. Activating immunity in the liver. II. IFN-β attenuates NK cell-dependent liver injury triggered by liver NKT cell activation. J Immunol. 2002; 168: 3763-3770.