Decellularized Human and Porcine Liver Matrix: Application for Graft Engineering.

Abstract

Liver transplantation is the only effective treatment for end stage liver disease, however, the number of good quality liver grafts is not sufficient to meet the need. Alternative approaches need to be explored to overcome this shortage, including the use of bioengineered hepatic tissue as transplantable grafts. Recent evidence from rodent models, suggests that decellularization of whole liver organs is feasible and provides an excellent scaffold for reseeding liver (stem) cells for graft engineering. However, there is ample experience with decellularization of larger livers from pigs or humans. The aim of this study is to develop an effective method for decellularization of porcine and human livers. Whole or partial porcine (n=3) and human livers that are rejected for transplantation (n=5) were cannulated via the portal vein and the hepatic artery and perfused (60 ml/min) with heparinized 0.9% NaCL to remove blood, followed by 4% Triton X-100 + 1% NH4OH to remove all cells and fatty acids present in the liver. This process takes 8 to 16 hours in which 4L detergent is recircutated and refreshed every 4 hours. In between recirculation rounds, 2L Triton-solution is perfused through the liver. After the decellularization procedure, the extracellular matrix (ECM) is rinsed with 0.9% NaCL and DNase to remove all remnants of cells and nucleases. The decelled matrix was analyzed for the absence of cells, RNA and DNA content and for the presence of matrix proteins. Histological analysis (H&E staining) showed that with the current method, virtually all nuclei were removed and that the elastin and collagen fibers (Weigert's RF and Picro-Sirius Red staining) was not affected by the decellularization process. We found a 93% ± 3% (average ± SD) reduction of RNA and a 47% ± 22% (average ± SD) reduction of DNA content. Preliminary results of re-seeding the ECM with green-fluorescent labeled mesenchymal stem cells (MSC) indicate that the matrix is not toxic for stem cells. The seeded MSC adhere to the matrix and spread. In conclusion, we established a method to make a-cellular ECM from whole porcine and whole and partial human livers. These decellularized matrices provide an excellent scaffold for reseeding liver cells, liver organoids and/or stem cells for graft engineering.