Preclinical Studies for Cell Transplantation: Isolation of Primate Fetal Hepatocytes, Their Cryopreservation, and Efficient Retroviral Transduction

ABSTRACT Fetal hepatocytes are an attractive target for in utero cellular transplantation. Their use could provide a very efficient way for implanting normal or transduced cells into the livers of affected fetuses. Marking cells with recombinant retroviruses is a powerful tool for evaluating the chimerism of grafted animals. The technique relies on the ex vivo transduction efficiency of the engrafted cells. We have isolated fetal primary hepatocytes from nonhuman primates. The cells were cultured and transduced with a retroviral vector carrying the Escherichia coli β-galactosidase gene. Optimal gene transfer efficiency was obtained 48–60 hr after plating and was as high as 90%. Cryopreservation had little effect on cell viability and infectivity: The viability of thawed hepatocytes remained high (75–85%) and the infection efficiency was identical to that of freshly isolated cells. Efficient ex vivo retroviral gene transfer into fetal hepatocytes provides an appropriate system for testing allogenic graftin...