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Background: Cell therapy, such as hepatocyte transplantation (HTx), is promising for the treatment of metabolic liver diseases or as a bridge to orthotopic liver transplantation in patients with fulminant liver failure. However, one of the limitations of this therapy is the shortage of donors. The present study aims to investigate whether the two-layer method (TLM) of cold preservation with oxygenation improves the viability and activity of hepatocytes from rat donation after cardiac death (DCD) donors compared with results obtained with the University of Wisconsin (UW) solution. Moreover, we evaluated the hepatocyte function after culture or transplantation into the spleen. Materials and Methods: We used male Sprague-Dawley rats for this study. The DCD model was induced by phrenotomy after injecting heparin. We assigned rats based on warm ischemia times of 15 and 30 min to groups S and L, respectively. Each group (n = 5) was then subdivided as follows: (1) group S: not preserved (S/N), preserved by TLM for 3 h (S/TLM3) and 12 h (S/TLM12), and in the UW solution for 3 h (S/UW3) and 12 h (S/UW12), and (2) group L: not preserved (L/N), preserved by TLM for 3 h (L/TLM3) and 12 h (L/TLM12), and in the UW solution for 3 h (L/UW3) and 12 h (L/UW12). The cell viability and function of isolated DCD hepatocytes were analyzed for culture or HTx into the spleen. Results: The viability and ATP levels of DCD hepatocytes significantly improved after TLM compared with the values after preservation in cold UW solution in group S/N (p < 0.059). The levels of albumin production and urea synthesis by hepatocytes after culture were significantly higher in groups S/TLM3 and S/TLM12 than in groups S/UW3 and S/UW12 (p < 0.05), respectively. Further, serum albumin levels after HTx were also markedly higher in groups S/TLM3 and S/TLM12 than in groups S/UW3 and S/UW12. The morphological features revealed that cultured and transplanted hepatocytes remained clearly viable and maintained an expression for specific hepatic function, such as the production of albumin and glycogen. Conclusion: This novel method of oxygenated cold preservation of DCD livers can expand the hepatocyte donor pool for HTx and establish a wider application of this developing technique.

Background: Pulmonary tissue is protected from fluid leakage by an endothelial structural barrier, the glycocalyx (GCX). Once this barrier is disrupted, pulmonary edema rapidly develops. The depletion of the GCX is associated with leukocyte accumulation in the pulmonary vasculature, possibly causing the endothelial cells to become hyperpermeable. Whether neutrophils or macrophages are responsible for the development of pulmonary edema remains controversial. We used a mouse model of pulmonary edema induced by cognate anti-MHC antibody to explore the pathophysiological mechanism of pulmonary edema by examining the role of responsive neutrophils and macrophages and their interactions with the GCX.Methods: Anti-MHC class I antibody was administered intravenously to male BALB/c mice to induce pulmonary edema. Pulmonary edema was evaluated by measuring the wet-to-dry weight ratio of the lungs. Changes in the GCX were evaluated by electron microscopy and measurements of the serum level of soluble syndecan-1, a major GCX component. Heparin sulphate was administered to examine its protective effect on the GCX. Macrophages were depleted using clodronate to examine their role in the development of pulmonary edema. Results: The GCX of the pulmonary vascular endothelium degraded after the administration of an anti-MHC class I antibody, accompanied by an increase in the serum syndecan-1 and heparan sulfate levels. Macrophage depletion inhibited the development of pulmonary edema, and the administration of supplemental heparin, an inhibitor of heparan sulfate-degrading enzymes, suppressed the pulmonary edema. Conclusions: We demonstrated that the GCX is degraded in a mouse model of pulmonary edema induced by anti-MHC class I antibody. Macrophage depletion suppressed the development of the pulmonary edema. These results suggest that macrophages (and/or monocytes) may play a key role in pulmonary edema. Heparin inhibited both the degradation of the GCX in the pulmonary vascular endothelium and pulmonary edema. Our study may suggest an interventional mechanism for inhibiting pulmonary edema.