Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination.

Aim: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40Tag).

Methods: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets.

An improved purification approach with high cell viability and low cell loss for cryopreserved hepatocytes.

A modified purification procedure is described for effectively eliminating dead cells after hepatocyte cryopreservation. Isolated hepatocytes from six pig tissue samples were cryopreserved in liquid nitrogen for 2 weeks. After thawing, we developed a pre-incubation step prior to gradient centrifugation.