Percoll density gradient centrifugation media

Rat hepatocytes were isolated by the two-step collagenase perfusion technique as described previously^{28 (link)}. First, Ca²⁺-free Hanks’ balanced salt solution (HBSS) (Sigma-Aldrich, St. Louis, MO, USA) containing ethylene glycol tetraacetic acid was perfused through the portal vein at a rate of 14 ml/min for 5 min. Second, Ca²⁺-containing HBSS with 0.5 mg/ml of collagenase (Sigma type V; Sigma-Aldrich) was perfused via the same route at the same rate. The isolated cells were suspended in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) containing 10% fetal bovine serum and 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid. The cells were then filtered through a #150 mesh (Ikemoto Scientific Technology, Tokyo, Japan) and purified by gradient centrifugation (50 g, 2 min) at 4°C. Gradient centrifugation (50 g, 20 min) at 4°C was performed again using Percoll density gradient centrifugation media (physical form: Colloidal solution of silica coated with polyvinylpyrrolidone, density max: 1.135 g/ml osmolality: <25 mOsm/kg, viscosity max: 15cP, conductivity: <100 mS/m, pH range: 8.5–9.5) (GE Healthcare Biosciences, Pittsburgh, PA, USA) to obtain a highly purified cell population. The hepatocyte viability was evaluated by a trypan blue exclusion assay. We transplanted 1.0 × 10⁷ hepatocytes with a viability exceeding 90%.

Rat hepatocytes were isolated by the 2-step collagenase perfusion technique as described previously.^{24 (link)} First, Ca2⁺-free Hanks’ balanced salt solution (Sigma-Aldrich, St. Louis, MO) containing ethylene glycol tetraacetic acid was perfused through the portal vein at a rate of 14 mL/min for 5 minutes. Second, Ca2⁺-containing Hanks balanced salt solution with 0.5 mg/mL of collagenase (Sigma type V) (Sigma-Aldrich) was perfused via the same route at the same rate. The isolated cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) containing 10% fetal bovine serum (FBS) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The cells were then filtered through a 150 mesh (Ikemoto Scientific Technology, Tokyo, Japan) and purified by gradient centrifugation (50g, 2 minutes). Next, gradient centrifugation (50g, 20 minutes) was performed again using Percoll density gradient centrifugation media (GE Healthcare Biosciences, Pittsburgh, PA) to obtain a highly purified cell population. The yield and viability of the hepatocyte preparations were assessed immediately after isolation using trypan blue exclusion (TBE).

Hepatocyte isolation was performed using a 2-step collagenase perfusion method, as previously described.^{10 (link)} The isolated cells were suspended in Dulbecco's Modified Eagle’s Medium (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (Equitech-Bio, Inc, Kerrville, TX) and HEPES (Gibco, Thermo Fisher Scientific Inc, Waltham, MA). Density gradient centrifugation (50G, 20 min, 4 °C) was performed using Percoll density gradient centrifugation media (GE Healthcare Biosciences, Pittsburgh, PA) to obtain a highly purified cell population. Hepatocyte viability was measured by the trypan blue exclusion assay, and hepatocytes with ≥90% viability were used in the following experiments.