AIM To analyze retrospectively a 5-year experience of human hepatocyte isolation from resected liver tissues with benign disease.METHODS We established a method of modified four-step retrograde perfusion to isolate primary human hepatocytes. Samples were collected from the resected livers of patients with intrahepatic duct calculi(n = 7) and liver hemangioma(n = 17). Only the samples weighing ≥ 15 g were considered suitable for hepatocyte isolation. By using the standard trypan blue exclusion technique, hepatocyte viability and yield were immediately determined after isolation.RESULTS Twenty-four liver specimens, weighing 15-42 g, were immediately taken from the margin of the removed samples and transferred to the laboratory for hepatocyte isolation. Warm ischemia time was 5-35 min and cold ischemia time was 15-45 min. For the 7 samples of intrahepatic duct calculi, the method resulted in a hepatocyte yield of 3.49 ± 2.31 × 10~6 hepatocytes/g liver, with 76.4% ± 10.7% viability. The 17 samples of liver hemangioma had significantly higher yield of cells(5.4 ± 1.71 × 10~6 cells/g vs 3.49 ± 2.31 × 10~6 cells/g, P < 0.05) than the samples of intrahepatic duct calculi. However, there seems to be no clear difference in cell viability(80.3% ± 9.67% vs 76.4% ± 10.7%, P > 0.05). We obtained a cell yield of 5.31 ± 1.87 × 10~6 hepatocytes/g liver when the samples weighed > 20 g. However, for the tissues weighing ≤ 20 g, a reduction in yield was found(3.08 ± 1.86 × 10~6 cells/g vs 5.31 ± 1.87 × 10~6 cells/g, P < 0.05).CONCLUSION Benign diseased livers are valuable sources for largenumber hepatocyte isolation. Our study represents the largest number of primary human hepatocytes isolated from resected specimens from patients with benign liver disease. We evaluated the effect of donor liver characteristics on cell isolation, and we found that samples of liver hemangioma can provide better results than intrahepatic duct calculi, in terms of cell yield. Furthermore, the size of the tissues can affect the outcome of hepatocyte isolation. AIM To analyze retrospectively a 5-year experience of human hepatocyte isolation from resected liver tissues with benign disease. METHODS We established a method of modified four-step retrograde perfusion to isolate primary human hepatocytes. Samples were collected from the resected livers of patients with intrahepatic Only the samples weighing ≥ 15 g were considered suitable for hepatocyte isolation. By using the standard trypan blue exclusion technique, hepatocyte viability and yield were immediately determined after isolation .RESULTS Twenty-four liver specimens, weighing 15-42 g, were immediately taken from the margin of the removed samples and transferred to the laboratory for hepatocyte isolation. Warm ischemia time was 5-35 min and cold ischemia time was 15-45 min. For the 7 samples of intrahepatic duct calculi, the method resulted in a hepatocyte yield of 3.49 ± 2.31 × 10 6 hepatocytes / g liver, with 76.4% ± 10.7% viability. T he 17 samples of liver hemangioma had significantly higher yield of cells (5.4 ± 1.71 × 10 ~ 6 cells / g vs 3.49 ± 2.31 × 10 ~ 6 cells / g, P <0.05) than those samples of intrahepatic duct calculi. However, there seemed to have no clear difference in cell viability (80.3% ± 9.67% vs. 76.4% ± 10.7%, P> 0.05). We obtained a cell yield of 5.31 ± 1.87 × 10 ~ 6 hepatocytes / g liver when the samples weighed> 20 g. However, for the tissues weighing ≤ 20 g, a reduction in yield was found (3.08 ± 1.86 × 10 ~ 6 cells / g vs. 5.31 ± 1.87 × 10 ~ 6 cells / g, P < are valuable sources for largenumber hepatocyte isolation. Our study represents the largest number of primary human hepatocytes isolated from resected specimens from patients with benign liver disease. We evaluated the effect of donor liver characteristics on cell isolation, and we found that samples of liver hemangioma can provide better results than intrahepatic duct calculi, in terms of cell yield. Furthermor e, the size of thetissues can affect the outcome of hepatocyte isolation.