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Neohybrid Liver Graft - a novel concept of in vivo tissue-engineering

Martina T. Mogl¹, Susanne Rohn¹, Henriette Riedel¹, Jan Schroeder¹, Nils Billecke¹, Nathanael Raschzok¹, Lydia Brusendorf¹, Dietrich Polenz², Kerstin Nehls¹, Birgit Sawitzki², Igor M. Sauer¹

¹Department of General, Visceral and Transplant Surgery, Experimental Surgery and Regenerative Medicine, Charité Campus Virchow-Klinikum, university medicine; ²Berlin Brandenburg Center for Regenerative Therapies, Charité University Medicine, Berlin, Germany

Introduction: Liver transplantation is an effective therapy for end-stage liver disease, but life-long immunosuppression may lead to serious complications. Hepatocyte transplantation alone still has not reached long-lasting effects, mainly due to hepatocyte failure over time. Here, we investigated whether transplantation of syngeneic hepatocytes into allogeneic liver grafts may be used as a novel concept to improve tolerance induction.

Methods: Male Lewis rats were pretreated with 2-acetaminophen and partial hepatectomy to induce proliferation of hepatocytes and progenitor cells, especially oval cells. Hepatocytes and progenitor cells were isolated in a modified two-step procedure according to Seglen with density gradient separation of progenitor cells. Cells were transplanted into female Lewis rats that had undergone allogeneic liver transplantation. Livers were retrieved from female Dark Agouti rats pretreated with Retrorsin to facilitate cell engraftment. 5 x 10_6 Hepatocytes and progenitor cells were transplanted via the spleen, either immediately following organ transplantation, or up to 24hrs after organ transplantation after full recovery of recipient rats. Transplanted Lewis rats received continuous immunosuppression with Cyclosporin A and were sacrificed at days 8, 15, 30 and 90. Liver cell integrity was evaluated measuring transaminase levels. Detection of transplanted male cells was carried out by FiSH-typing of y-chromosomes and additional immunhistochemical staining of OV-6, CK18, CK19 and BrdU.

Results: Animals surviving the first 3 days after combined hepatocyte and liver transplantation showed stable liver function until they were sacrificed. FiSH-typing revealed moderate to good engraftment of transplanted male cells at all time-points mainly close to portal tracts. Counting of male cells/vision field (200x) showed a proportion between 13% and 38% of male cells per total cell count. So far no statistically significant difference between time-points could be detected (n=4/time-point).

Conclusion: In order to establish the new concept of a neohybrid liver graft we could proof a relevant proportion of engrafted cells up to 90 days after combined liver and hepatocyte transplantation. Cell transplantation via the spleen seems to be a safe and reliable technique, even after major surgery like liver transplantation. Influence of progenitor cells on engraftment and proliferation of transplanted hepatocytes are currently under evaluation, especially with respect to minimization of immunosuppression and induction of tolerance.