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The basic idea of the Neo-Hybrid Liver Graft project is to combine the two therapeutic options of liver transplantation and liver cell transplantation. The final objective is to develop a new concept of in vivo tissue engeneering of the liver by performing a cell transplantation of autologous hepatocytes after an orthotopic liver transplantation has been conducted. This is anticipated to induce a partial or complete absence of detrimental immune response towards the allogeneic liver graft, called operational tolerance, and consequently reduce the need for immunosuppressants. For this purpose functional hepatocytes and hepatic progenitor cells from explanted, diseased human livers were isolated and examined with regard to their suitability for cell transplantation. Hepatocyte isolations from sections of 23 explanted diseased human livers were carried out and compared to the results of 14 isolations from normal liver tissue after partial hepatectomy. The hepatocytes were cultured and examined for their viability and metabolic activity. Additionally, the cells were stored at 4°C for up to 48 hours to analyze stability during cold storage. The non parenchymal cell fraction was characterized by flow cytometry and two methods for liver progenitor cell enrichment were tested. Furthermore, a new mouse model of liver cell transplantation was developed with the intent to later test the repopulative capacity of hepatocytes from diseased, explanted liver tissue. Despite of a clinically confirmed liver failure as an indication of orthotopic liver transplantation, fully functional hepatocytes from explanted liver tissue could be successfully isolated and cultivated. Moreover, no significant difference to the isolation outcome of healthy liver tissue could be found. In hepatocyte cultures only minor differences in viability and metabolic activity could be observed between cells from diseased and healthy liver tissue. Indeed, hepatocytes from diseased, explanted liver tissue appeared to be more robust in long term culture. The cold storage of hepatocytes for 24 and 48 hours resulted in acceptable losses of cell viability, which differed only slightly between the two tested storage media ChillProtec® and ChillProtec® plus. Again, hepatocytes from diseased liver tended to be more robust than those from healthy liver tissue. The non parenchymal cell fractions were initially tested for the cell surface protein EpCAM, a known marker of hepatic progenitor cells, and for the leucocyte marker CD45. The non parenchymal cell fraction of diseased, explanted liver tissue contained more EpCAM-positive cells than those obtained from partial liver resections. However, these differences were not statistically significant. The majority of EpCAM-positive cells turned out to be also positive for the surface marker CD133/1, confirming the phenotype of progenitor cells. Making up about half of the non parenchymal cells the EpCAM- and CD45-negative cell fraction of diseased, explanted livers contained significantly more CD90-positive cells than those of partial liver resections. These cells also represented potential progenitors of hepato- or cholangiocytes as CD90 has been repeatedly described as a marker of hepatic progenitor cells. Subsequent enrichment of these potential progenitor cells for immediate cell transplantation by the means of OptiPrep™ density gradient centrifugation or magnetic-activated cell sorting (MACS®) proved to be inappropriate.
The new mouse model for liver cell transplantation, which had been optimized to fit this project, showed successful engraftment of human hepatocytes isolated from normal tissue of partial liver resections. Above all, the viability of cells at the time of transplantation turned out to play an important role for successful engraftment. Potential sources of error during execution and analysis of the model have been assessed in order to optimize its scheduled application with hepatocytes from diseased, explanted liver. Altogether, the results presented here support the hypothesis that hepatocytes from diseased, explanted human livers represent an adequate cell source for hepatocyte transplantation. Moreover, there was evidence that a sufficient number of hepatic progenitor cells can be isolated from diseased liver for cell transplantation. Both cell types now need to be tested for their repopulative capacity using the newly developed mouse model. The Neo-Hybrid Liver Graft project aims to extend survival time and improve quality of life of people undergoing orthotopic liver transplantation. The hereby presented results support the feasibility of the concept.