Introduction: Although liver transplantation is the only curative and established treatment for end-stage liver disease, patients that could have benefitted from transplantation are restricted by the severe donor organ shortage. The transplantable whole-liver engineering using decellularization technique is one of the approaches to resolve this problem, however, coagulation in the decellularized liver stopped the blood flow and there was no study to achieve clinical relevant recellularized liver grafts. Inappropriate cell distribution and coagulation with blood perfusion were tasks to be solved for practical use. The current study proposed a method substituted for transplantation as clinical application of recellularized liver graft, reproducing appropriate cell distribution.
Methods: The Lewis male rat livers were used for generating the whole-organ decellularized scaffolds and cell isolation. Isolated primary hepatocytes and liver sinusoidal endothelial cells (LSECs) were recellularized via bile duct and portal vein, respectively, and perfusion culture was performed. Histological analysis was evaluated for cell distribution and function in recellularized liver. After perfusion culture, we put our engineered liver graft into the extracorporeal perfusion system using an alive Lewis rat.
Results: Histological analysis revealed appropriate distribution of hepatocytes into the parenchymal space without intra-portal thrombosis, resulting in LSECs attachment along the portal vein, maintaining characteristic morphology and phenotype. Platelet deposition evaluated by measuring fluorescent intensity of integrin αIIb+ was significantly decreased in hepatocytes and LSECs co-seeded group than in hepatocyte alone seeded group (p < 0.001). Blood clotting did not stop the blood flow during 3 h extracorporeal perfusion.
Conclusion: Biliary duct seeding of parenchymal cells could allow endothelial cell attachment along the vasculature and did not obstruct blood flow into vessels. LSECs could maintain their morphology and phenotype, and possess anti-coagulation ability in engineered liver grafts. Extracorporeal perfusion might be one of the solutions for clinical use of recellularized liver graft. Further study is needed to investigate our fabricated liver graft could support liver function in the extracorporeal perfusion system.
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