Porcine Liver Decellularization Under Oscillating Pressure Conditions: A Technical Refinement to Improve the Homogeneity of the Decellularization Process

Abstract

Decellularization and recellularization of parenchymal organs may facilitate the generation of autologous functional liver organoids by repopulation of decellularized porcine liver matrices with induced liver cells. We present an accelerated (7 h overall perfusion time) and effective protocol for human-scale liver decellularization by pressure-controlled perfusion with 1% Triton X-100 and 1% sodium dodecyl sulfate via the hepatic artery (120 mmHg) and portal vein (60 mmHg). In addition, we analyzed the effect of oscillating pressure conditions on pig liver decellularization (n=19). The proprietary perfusion device used to generate these pressure conditions mimics intra-abdominal conditions during respiration to optimize microperfusion within livers and thus optimize the homogeneity of the decellularization process. The efficiency of perfusion decellularization was analyzed by macroscopic observation, histological staining (hematoxylin and eosin [H&E], Sirius red, and alcian blue), immunohistochemical staining (collagen IV, laminin, and fibronectin), and biochemical assessment (DNA, collagen, and glycosaminoglycans) of decellularized liver matrices. The integrity of the extracellular matrix (ECM) postdecellularization was visualized by corrosion casting and three-dimensional computed tomography scanning. We found that livers perfused under oscillating pressure conditions (P(+)) showed a more homogenous course of decellularization and contained less DNA compared with livers perfused without oscillating pressure conditions (P(-)). Microscopically, livers from the (P(-)) group showed remnant cell clusters, while no cells were found in livers from the (P(+)) group. The grade of disruption of the ECM was higher in livers from the (P(-)) group, although the perfusion rates and pressure did not significantly differ. Immunohistochemical staining revealed that important matrix components were still present after decellularization. Corrosion casting showed an intact vascular (portal vein and hepatic artery) and biliary framework. In summary, the presented protocol for pig liver decellularization is quick (7 h) and effective. The application of oscillating pressure conditions improves the homogeneity of perfusion and thus the outcome of the decellularization process.

Bioengineered Livers: A New Tool for Drug Testing and a Promising Solution to Meet the Growing Demand for Donor Organs

“…In translational studies, large animals such as pigs and sheep are preferred [25,26,27,28,29,30,31,32]. The use of pigs as organ donors is discussed controversially because of the immunological barrier due to the expression of galactose-1,3-alpha-galactose in porcine organs which may lead to hyperacute rejection of the repopulated organ.…”

“…However, the exclusive application of this detergent resulted in incomplete decellularization with nuclear material remaining in the scaffold. In most previous studies, a combination of these two detergents was applied [6,8,10,13,14,16,22,24,26,29,35,36]. As a result, no remaining cellular and nuclear material was detected, and the structure of the extracellular components was maintained (fig.…”

“…As the main route for the perfusion, the portal vein was used. Alternatively, the hepatic artery or the vena cava served as perfusion route [14,16,22,26]. To secure the outflow, the hepatic vessels like the hepatic veins and the vena cava inferior were severed before the perfusion started.…”

“…The newest approach was demonstrated in the recently published studies from Struecker et al [22,26]. They introduced perfusion under physiological oscillating pressure conditions via the hepatic artery and the portal vein, and applied this technique successfully in two animal models - rats and pigs.…”

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

“…These pressure conditions mimic the physiologic respiratory-dependent perfusion of the liver: in situ, the liver hangs under the copula of the diaphragm, whose movement during respiration has a direct impact on liver perfusion. Inspiration specifically leads to lowering of the diaphragm and squeezing of the liver, optimizing hepato-venous outflow, whereas expiration leads to elevation of the liver and lowering of the intraabdominal pressure to optimize portal-venous inflow 15 .…”