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331h

Making of a New Liver Using Decellularized Livers as Scaffolds

Basak E. Uygun1, Alejandro Soto-Gutierrez1, Carley Shulman1, Korkut Uygun2, François Berthiaume3, and Martin Yarmush4. (1) Surgery, Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, 51 Blossom Street, Boston, MA 02114, (2) Center for Engineering in Medicine, Harvard Med School, 51 Blossom Street, Boston, MA 02114, (3) Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, 51 Blossom Street, Boston, MA 02114, (4) Research, Center for Engineering in Medicine, 51 Blossom Street, Boston, MA 02114

Orthotopic liver transplantation is the only clinically proven effective treatment for end-stage liver disease and there are currently ~100,000 patients on the organ transplant waiting list in the US, a number that far exceeds the supply of available organs, and that continues to grow ~5% each year. One of the most promising strategies that is being explored as means for addressing this critical shortage is constriction of bioartificial tissue and organs (which aims to manufacture tissue and organ analogues in vitro). In this study, we aimed to reform liver tissue using perfusion decellularized rat livers as scaffolds. Rat livers were harvested from cadaveric donors and decellularized by perfusion with 1% sodium dodecyl sulfate. The removal of the cellular component from the liver was confirmed by hematoxylin and eosin staining of histology sections of the decellularized specimen. The decellularized livers were recellularized with adult primary hepatocytes (100 million cells, 90% viability) isolated by perfusion incubation over 60 minutes and the recellularized livers were cultured in organ perfusion system for 24 hours. At the end of the seeding period, it was found that 80 million cells were retained in the organ and cell viability was approximately 70%. Viability of the cells during perfusion culture was monitored by measuring lactate dehydrogenase activity in the perfusion medium. Hepatic metabolic function was measured by analysis for urea and albumin secretion in the medium. At the end of the 24 hr perfusion culture, histology analysis of the recellularized liver samples revealed that cells were distributed uniformly around the openings which used to constitute liver vasculature. Hepatocytes were found to be positive for glycogen after periodic acid-Schiff staining. There were very minimal apoptotic cells as confirmed by TUNEL staining. The results showed that hepatocytes successfully grafted in the decellularized livers using organ perfusion system. Future work will include demonstration of long-term function of hepatocytes after repopulating the decellularized liver.