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 SV40T. SV40T 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.