(516f) An Engineered Liver Graft With Improved Blood Compatibility and Prolonged Survival

Introduction: The only definitive treatment for end-stage liver disease is orthotopic liver transplantation and it is limited by the shortage of available donor organs. To address the issue, we developed a novel method to prepare transplantable liver grafts using perfusion decellularized liver matrix (DLM). While we successfully showed that the engineered liver grafts were functional in vitro and could be transplanted in rats, the post-transplantation survival time of the recipient animals was significantly short (~8 h) due to complications related to the high thrombogenicity of the grafts. An ideal engineered liver graft should enable undisrupted blood circulation through the graft in order to allow for its therapeutic testing in a liver failure model. Therefore, we hypothesized that the deposition of an anti-coagulant, heparin, on the DLM scaffold surface will render the DLM blood compatible, and consequently will prevent blood clotting when exposed to blood upon transplantation and prolong the post transplantation survival time of the recipient animal.

Materials and Methods: Heparin was immobilized on the interior surface of DLM via layer-by-layer (LbL) deposition technique. Heparin and poly(diallydimethly ammoniumchloride) were used as negatively charged and positively charged polyelectrolytes, respectively. Four different conditions of heparin LbL films were deposited at two different bilayers of heparin (4 and 8 BL) and at two different concentrations of heparin (2 g/L and 4 g/L). The heparin LbL film was verified via toluidine blue staining and was quantified by hexosamine assay. The ability of the heparinized DLM (hDLM) to prevent blood coagulation was tested via 2-h ex vivo diluted blood perfusion. The hDLM scaffolds were recellularized with primary hepatocytes and the hepatic functions of recellularized hDLM were observed over 5-day culture period. The recellularized hDLM scaffolds were perfused with diluted blood for 24-h ex vivo to evaluate the extent of blood coagulation prior to transplantation. The engineered liver grafts that showed the maintenance of hepatic functions and the reduced blood coagulation in vitrowere transplanted via inferior vena cava anastomosis with the use of cuff technique. Animal survival time was monitored over 3 weeks and the transplanted liver grafts were harvested 1d post-transplantation for histological assessment.

Results and Discussion: Four different types of hDLM scaffolds were prepared to verify heparin immobilization via toluidine blue staining. The amount of heparin deposited ranged 20-80 mg depending on the number of heparin bilayers and the concentration of heparin used. The 2-h ex vivo blood perfusion showed that the time and extent of blood clot formation varied depending on the level of heparin in the scaffolds. The hDLM deposited with 8 bilayers of heparin at 4 g/L showed the most significant reduction in the blood clot formation. Albumin and urea production of the recellularized hDLM scaffolds were comparable to that observed in unheparinized DLM over 5d of in vitro perfusion culture, indicating heparin deposition has no effect on the maintenance of hepatic functions over the culture time. As a result of 24-h ex vivodiluted blood perfusion, the heparinized liver grafts exhibited reduced blood coagulation as observed by reduced flow resistance when compared to unheparinized liver graft. Animals that received the engineered liver grafts as auxiliary grafts survived for longer than 3 weeks. Histological analyses showed the significant prevention of blood coagulation in the heparinized liver grafts post-transplantation.

Conclusions: The engineered liver grafts with heparin layers exhibited enhanced survival by reducing blood clot formation post-transplantation. The generation of transplantable and functional engineered liver grafts that can serve as a reliable alternative for liver transplantation will offer a great potential for developing treatment of liver diseases.