Reversible immortalization of human hepatocytes mediated by retroviral transfer and site-specific recombination

AIM: To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications.METHODS: We successfully isolated primary human hepatocytes from surgically resected liver tissue taken from a patient with liver hemangiomas. The freshly isolated cells were then immortalized with retroviral vector SSR#69 expressing simian virus 40 large T antigen(SV40T) and hygromycin-resistance genes flanked by paired loxP recombination targets.RESULTS: The freshly isolated hepatocytes with high viability(85%) were successfully immortalized using retroviral gene transfer of SV40 T. SV40 T in the immortalized cells was then excised by Cre/loxP site-specific recombination. This cell population exhibited the characteristics of differentiated hepatocytes.CONCLUSION: We successfully established reversibly immortalized human hepatocytes, which will provide an unlimited supply of cells for practical applications.

Surgical treatment for uncinate process carcinoma of the pancreas

Objectives: To analyze the clinical features of unci- nate process carcinoma of the pancreas and to im- prove the resection rate. Methods: From January 1990 to June 1999, 10 pa- tients with pancreas uncinate process carcinoma re- ceived Whipple's operation. Portal vein (PV) resec- tion and reanastomosis were performed in 5 patients, and the resected length varied from 2.0 to 4.2 cm. Two patients underwent PV lateral wall partial resec- tion. Results: Among the 7 patients undergoing PV resec- tion, 1 died of hepatic failure 3 days after operation. One patient suffered from postoperative chylous asci- tes. These 6 patients survived 13 to 29 months post- operatively. Among the 3 patients without PV resec- tion, 2 survived 13 months and 14 months respective- ly. One patient was alive by the end of follow-up for 11. 5 months postoperatively. Conclusion: Although uncinate process carcinoma of the pancreas has a tendency to invade the adjacent PV and superior mesentery vein, it should not be simply regarded as a contraindication of radical resection.

Hepatocyte isolation from resected benign tissues: Results of a 5-year experience

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.