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 grafting and for modifying immunogenicity of engrafted cells. These results open up new perspectives for cell transplantation through cell banking.

Overview summary

The first goal of this study was to isolate nonhuman primate fetal hepatocytes efficiently. The culture conditions that provided the most efficient retroviral transduction were identified. Fetal hepatocytes were shown to be very sensitive to retroviruses, with 90% of cells being transduced. Cryopreservation had little or no effect on cell viability and infectivity. These results open up the possibility of cell banking.