Isolation of Mouse Primary Hepatic Stellate Cells

HSCs were isolated as described previously (22). Briefly, mice were perfused in situ through the portal vein with 1x EGTA [5.4 mM KCl, 0.44 mM KH₂PO₄, 140 mM NaCl, 0.34 mM Na₂HPO₄, 0.5 mM EGTA, 25 mM Tricine, and 1% penicillin/streptomycin (PS); pH 7.2] at a rate of 1.4 ml/min, followed by perfusion with 0.075% collagenase type I in HBSS at 37°C. Liver tissues were collected into digestion solution and homogenized. The tissue was incubated for 20 min at 37°C with shaking (100 rpm). The cell suspension was filtered through a 70 μm nylon cell strainer (BD Falcon) and centrifuged at 500 rpm for 5 min at room temperature to separate the hepatocytes. The supernatant was collected and centrifuged at 1,600 rpm at 4°C for 10 min, and the cell pellet was collected, resuspended in HBSS, and centrifuged again. Gradient solutions were prepared with Opti-Prep (40, 20, and 11.5%). The pellet was resuspended in 20% Opti-Prep (Sigma-Aldrich), slowly overlaid with 11.5% Opti-Prep and 0% Opti-Prep (HBSS), and centrifuged at 3,000 rpm at 4°C for 17 min without the brake. HSCs were collected from the interface of the 0 and 11.5% Opti-Prep layers. Freshly isolated primary HSCs were washed with HBSS, resuspended in media [RPMI 1,640 medium with 10% fetal bovine serum (FBS) and 1% antibiotics], and counted.