Online oxygen monitoring using integrated inkjet-printed sensors in a liver-on-a-chip system.

The demand for real-time monitoring of cell functions and cell conditions has dramatically increased with the emergence of organ-on-a-chip (OOC) systems. However, the incorporation of co-cultures and microfluidic channels in OOC systems increases their biological complexity and therefore makes the analysis and monitoring of analytical parameters inside the device more difficult. In this work, we present an approach to integrate multiple sensors in an extremely thin, porous and delicate membrane inside a liver-on-a-chip device. Specifically, three electrochemical dissolved oxygen (DO) sensors were inkjet-printed along the microfluidic channel allowing local online monitoring of oxygen concentrations. This approach demonstrates the existence of an oxygen gradient up to 17.5% for rat hepatocytes and 32.5% for human hepatocytes along the bottom channel. Such gradients are considered crucial for the appearance of zonation of the liver. Inkjet printing (IJP) was the selected technology as it allows drop on demand material deposition compatible with delicate substrates, as used in this study, which cannot withstand temperatures higher than 130 °C. For the deposition of uniform gold and silver conductive inks on the porous membrane, a primer layer using SU-8 dielectric material was used to seal the porosity of the membrane at defined areas, with the aim of building a uniform sensor device. As a proof-of-concept, experiments with cell cultures of primary human and rat hepatocytes were performed, and oxygen consumption rate was stimulated with carbonyl-cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), accelerating the basal respiration of 0.23 ± 0.07 nmol s-1/106 cells up to 5.95 ± 0.67 nmol s-1/106 cells s for rat cells and the basal respiration of 0.17 ± 0.10 nmol s-1/106 cells by up to 10.62 ± 1.15 nmol s-1/106 cells for human cells, with higher oxygen consumption of the cells seeded at the outflow zone. These results demonstrate that the approach of printing sensors inside an OOC has tremendous potential because IJP is a feasible technique for the integration of different sensors for evaluating metabolic activity of cells, and overcomes one of the major challenges still remaining on how to tap the full potential of OOC systems.

The Effect of High-Mobility Group Box 1 in Rat Steatotic and Nonsteatotic Liver Transplantation From Donors After Brain Death.

High-mobility group box 1 (HMGB1) has been described in different inflammatory disorders, and the deleterious effects of brain death (BD) may counteract the protection conferred by ischemic preconditioning (IP), the only surgical strategy that is being applied in clinical liver transplantation. Our study examined how HMGB1 may affect preconditioned and unpreconditioned steatotic and nonsteatotic liver grafts from donors after BD (DBDs) for transplantation. HMGB1 was pharmacologically modulated in liver grafts from DBDs, and HMGB1-underlying mechanisms were characterized. We found that BD decreased HMGB1 in preconditioned and unpreconditioned livers and was associated with inflammation and damage. Exogenous HMGB1 in DBDs activates phosphoinositide-3-kinase and Akt and reduces hepatic inflammation and damage, increasing the survival of recipients. Combination of IP and exogenous HMGB1 shows additional benefits compared with HMGB1 alone. This study provides new mechanistic insights into the pathophysiology of BD-derived liver graft damage and contributes to the development of novel and efficient strategies to ultimately improve liver graft quality.

Brain death and marginal grafts in liver transplantation.

It is well known that most organs for transplantation are currently procured from brain-dead donors; however, the presence of brain death is an important risk factor in liver transplantation. In addition, one of the mechanisms to avoid the shortage of liver grafts for transplant is the use of marginal livers, which may show higher risk of primary non-function or initial poor function. To our knowledge, very few reviews have focused in the field of liver transplantation using brain-dead donors; moreover, reviews that focused on both brain death and marginal grafts in liver transplantation, both being key risk factors in clinical practice, have not been published elsewhere. The present review aims to describe the recent findings and the state-of-the-art knowledge regarding the pathophysiological changes occurring during brain death, their effects on marginal liver grafts and summarize the more controversial topics of this pathology. We also review the therapeutic strategies designed to date to reduce the detrimental effects of brain death in both marginal and optimal livers, attempting to explain why such strategies have not solved the clinical problem of liver transplantation.

Somatostatin therapy protects porcine livers in small-for-size liver transplantation.

Small-for-size (SFS) injury occurs in partial liver transplantation due to several factors, including excessive portal inflow and insufficient intragraft responses. We aim to determine the role somatostatin plays in reducing portal hyperperfusion and preventing the cascade of deleterious events produced in small grafts. A porcine model of 20% liver transplantation is performed. Perioperatively treated recipients receive somatostatin and untreated controls standard intravenous fluids. Recipients are followed for up to 5 days. In vitro studies are also performed to determine direct protective effects of somatostatin on hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC). At reperfusion, portal vein flow (PVF) per gram of tissue increased fourfold in untreated animals versus approximately threefold among treated recipients (p = 0.033). Postoperatively, markers of hepatocellular, SEC and HSC injury were improved among treated animals. Hepatic regeneration occurred in a slower but more orderly fashion among treated grafts; functional recovery was also significantly better. In vitro studies revealed that somatostatin directly reduces HSC activation, though no direct effect on SEC was found. In SFS transplantation, somatostatin reduces PVF and protects SEC in the critical postreperfusion period. Somatostatin also exerts a direct cytoprotective effect on HSC, independent of changes in PVF.

Superoxide dismutase gene transfer reduces portal pressure in CCl4 cirrhotic rats with portal hypertension.

BACKGROUND: Increased intrahepatic vascular tone in cirrhosis has been attributed to a decrease of hepatic nitric oxide (NO) secondary to disturbances in the post-translational regulation of the enzyme eNOS. NO scavenging by superoxide (O(2)(-)) further contributes to a reduction of NO bioavailability in cirrhotic livers. AIM: To investigate whether removing increased O(2)(-) levels could be a new therapeutic strategy to increase intrahepatic NO, improve endothelial dysfunction and reduce portal pressure in cirrhotic rats with portal hypertension. METHODS: Adenoviral vectors expressing extracellular superoxide dismutase (SOD) (AdECSOD) or beta-galactosidase (Adbetagal) were injected intravenously in control and CCl(4)-induced cirrhotic rats. After 3 days, liver O(2)(-) levels were determined by dihydroethidium staining, NO bioavailability by hepatic cGMP levels, nitrotyrosinated proteins by immunohistochemistry and western blot, and endothelial function by responses to acetylcholine in perfused rat livers. Mean arterial pressure (MAP) and portal pressure were evaluated in vivo. RESULTS: Transfection of cirrhotic livers with AdECSOD produced a significant reduction in O(2)(-) levels, a significant increase in hepatic cGMP, and a decrease in liver nitrotyrosinated proteins which were associated with a significant improvement in the endothelium-dependent vasodilatation to acetylcholine. In addition, in cirrhotic livers AdECSOD transfection produced a significant reduction in portal pressure (17.3 (SD 2) mm Hg vs 15 (SD 1.6) mm Hg; p<0.05) without significant changes in MAP. In control rats, AdECSOD transfection prevents the increase in portal perfusion pressure promoted by an ROS-generating system. CONCLUSIONS: In cirrhotic rats, reduction of O(2)(-) by AdECSOD increases NO bioavailability, improves intrahepatic endothelial function and reduces portal pressure. These findings suggest that scavenging of O(2)(-) might be a new therapeutic strategy in the management of portal hypertension.