Interspecies difference in liver-specific functions and biotransformation of testosterone of primary rat, porcine and human hepatocyte in an organotypical sandwich culture.

Interspecies difference is an important issue in toxicology research. We compared the potential in vitro metabolism of human, porcine and rat hepatocytes over 2 weeks in culture in an organotypical culture model which reflects the in vivo situation. All three species show similar LDH-rates. Albumin measurements showed that rat cells are about twice as active as human and porcine hepatocytes. The ethoxyresorufin-O-deethylase (EROD) activity of the rat hepatocytes is with about 14 microU/10(6)cells distinctly higher than those of porcine and human cells (1.8 and 0.5 microU/10(6)cells respectively), furthermore, the activity of the rat EROD increases slightly during the prolonged time in culture, whereas those of porcine and human enzymes slightly decrease. Concerning ethoxycoumarin-O-deethylase (ECOD), the enzyme activities are found to be in three different ranges where rat cells show the highest activity with 66 microU/10(6)cells, porcine hepatocytes exhibit an activity of about 23 microU/10(6)cells, and human activity is lowest with 0.7 microU/10(6)cells. All three species show a similar decreasing trend of ECOD during the period of study. Regarding the biotransformation of testosterone, human and porcine liver cells form three major metabolites whereas rat cells form a mixture of all measured metabolites. Hence, in vitro metabolism using porcine hepatocytes would be much more scientific sense than one using rat hepatocytes since the metabolic pathways are much closer to human metabolism.

Detoxification and endothelialization of glutaraldehyde-fixed bovine pericardium with titanium coating: a new technology for cardiovascular tissue engineering.

BACKGROUND: Endothelial cell seeding of glutardialdehyde-fixed biological heart valves is hypothesized to improve biocompatibility and durability; however, the toxicity of glutardialdehyde prevents its use as a biological coating. Therefore, different detoxification strategies are applied, including surface coating with titanium, before in vitro endothelialization of glutaraldehyde-fixed bovine pericardium as the base material for prosthetic heart valves. METHODS AND RESULTS: Bovine pericardium was fixed with 0.25% glutardialdehyde. Detoxification was performed with citric acid, aldehyde dehydrogenase, and plasma deposition with titanium at low temperatures of 30 degrees C to 35 degrees C. Toxic glutaraldehyde ligands were quantified photometrically, and the vitality of seeded cells was tested to validate detoxification methods. Detoxification agents and titanium coating were applied before seeding with human endothelial cells. Endothelial cells were visualized by electron microscopic surface scanning. To evaluate cell adhesion, shear stress was applied by a flow of 5 L/min over 24 hours. Compared with untreated glutaraldehyde-fixed samples, treatment with the different agents reduced free aldehyde groups gradually (citric acid 5% < citric acid 10% < titanium < aldehyde dehydrogenase). A combination of citric acid 10%, aldehyde dehydrogenase, and titanium coating resulted in a reduction of free aldehyde ligands to 17.3+/-4.6% (P < or = 0.05) and demonstrated a vitality of seeded cells of 94+/-6.7% (P < or = 0.05). This procedure yielded a completely confluent layer of regular human endothelial cells (n=5). After application of shear stress for 24 hours on these endothelial layers, cell vitality was 81%. CONCLUSIONS: Titanium coating combined with chemical procedures yielded significant detoxification and complete endothelialization of conventional glutaraldehyde-fixed pericardium. This new technique might improve glutardialdehyde-fixed cardiovascular bioimplants for better biocompatibility and longer durability.

Development and characterization of a small-scale bioreactor based on a bioartificial hepatic culture model for predictive pharmacological in vitro screenings.

A vast majority of pharmacons are beset by possible interactions and side effects which have usually been tested in laboratory animals. However, better methods are needed to reduce the number of animal experiments and interspecies differences with respect to drug metabolism, as well as to provide a faster and more cost-effective way of analysis. These facts have led to the development of in vitro models based on isolated primary hepatocytes to better assess drug metabolism, interactions, and toxicity. A new small-scale bioreactor with the hepatic sandwich model and a gas-permeable membrane at the bottom allowing a definable oxygen exchange, has been constructed and compared with the conventional well plates. Compared to hepatocytes cultured in conventional systems, the cells exhibited a stronger liver-specific capacity and remained in a differentiated state in the small-scale bioreactor over a cultivation period of 17 days. This in vitro model could serve as a tool to predict the liver response to newly developed drugs.

Cultivation of primary porcine hepatocytes in an OXY-HFB for use as a bioartificial liver device.

Bioreactors being developed for bioartificial liver devices vary greatly in their construction. Until now, primary liver cells were cultivated either in sandwich configuration, as spheroids, or in special hollow fiber systems. Primary hepatocytes are demanding on their environment and have a high oxygen consumption. To get good results, optimal cultivation conditions are needed. The idea of the project was to investigate a new concept of an oxygenating hollow fiber bioreactor (OXY-HFB). The OXY-HFB should consist exclusively of oxygenating and internal heat exchange fibers to yield a simple and effective design. Primary liver cells were seeded on the surface of the fibers in the extrafiber space. Oxygen requirements and temperature control were supplied through the fibers. The culture medium was perfused through the extrafiber space and therefore brought into direct hepatocellular contact. The OXY-HFB concept offers different advantages. A high cell density of 2.5 x 10(7) cells/mL can be obtained. This results in a cell number of 2.5 x 10(9) liver cells per bioreactor. Furthermore, the OXY-HFB is easily handled because no incubator is required. To study the efficiency of this bioreactor technique, various parameters were investigated over a cultivation period of three weeks. These included urea synthesis, lactate formation, glucose elimination, albumin synthesis, oxygen level, and pH. Furthermore, the metabolites of diazepam were measured. The biochemical performance of the bioreactor remained stable over the investigated time period. These results demonstrate that porcine liver cells preserve their viability and primary metabolism in the OXY-HFB over the complete period of study.

Bioreactor developments for tissue engineering applications by the example of the bioartificial liver.

Tissue engineering is the application of the principles and methods of engineering and the life sciences towards the development of biological substitutes to restore, maintain or improve functions. It is an area which is emerging in importance worldwide. This article is to show the developments in tissue engineering research by the example of the bioartificial liver. As an alternative to liver transplantation, numerous researchers have been working towards the goal of development of a fully functional artificial liver. Liver support systems based on detoxification alone have proven ineffective because they cannot correct biochemical disorders. An effective artificial liver support system should be capable of carrying out the liver's essential processes, such as synthetic and metabolic functions, detoxification, and excretion. It should be capable of sustaining patients with fulminant hepatic failure and preparing patients for liver transplantation when a donor liver is not readily available. Although several hepatocyte-based liver support systems have been proposed, there is no current consensus on its eventual design configuration.