Upcyte® Hepatocytes show stable phenotype characteristics of human hepatocytes

Aims: The low availability of human liver tissue causes a limited supply of much needed primary human hepatocytes. To address this problem, Medicyte has developed a new technology which induces the ability of human hepatocytes to proliferate by transducing the cells with genes enabling cell proliferation. The aim of this work was to elucidate gene expression and metabolic characteristics of these specially modified liver cells called upcyte® Hepatocytes at different stages of expansion.

Material and Methods: Upcyte® Hepatocytes from different donors were cultured for three expansion stages, over ˜20 Population Doublings (PD). Expression levels of different genes responsible for urea synthesis, blood coagulation and protein synthesis were measured by RT-PCR. Cells were stimulated with ammonium chloride in presence or absence of ornithine to determine functional urea synthesis. To investigate further the efficiency of upcyte® Hepatocytes, the secretion of albumin was measured in media using an ELISA method. Additionally we compared the expression levels of phase-I-enzymes and receptors responsible for their activation after cultivation in 2D and 3D culture system.

Results: Upcyte® Hepatocytes grew in a linear fashion over time such that within two weeks, the cells proliferated from 6 million to about 120 million. RT-PCR results showed that these cells expressed all enzymes necessary for urea synthesis, protein synthesis and coagulation. The rate of urea production was lower in upcyte® than in primary hepatocytes but it was stable over the whole period of cell expansion (20 PDs). The two different culture systems indicated that the 3D culture system mimics in vivo conditions better than 2D cultivation.

Conclusion and Outlook: In conclusion, our findings suggest application of the upcyte® technology to human hepatocytes induces proliferation without marked loss of function in urea synthesis. Further elucidations will be necessary to test different metabolic characteristics of upcyte® Hepatocytes in order to use them for cell-based therapy.