Primary hepatocytes represent a well-accepted in vitro cell culture system for studies of drug metabolism, enzyme induction, transplantation, viral hepatitis, and hepatocyte regeneration. Recently, a multicentric research program has been initiated to optimize and standardize new in vitro systems with hepatocytes. In this article, we discuss five of these in vitro systems: hepatocytes in suspension, perifusion culture systems, liver slices, co-culture systems of hepatocytes with intestinal bacteria, and 96-well plate bioreactors. From a technical point of view, freshly isolated or cryopreserved hepatocytes in suspension represent a readily available and easy-to-handle in vitro system that can be used to characterize the metabolism of test substances. Hepatocytes in suspension correctly predict interspecies differences in drug metabolism, which is demonstrated with pantoprazole and propafenone. A limitation of the hepatocyte suspensions is the length of the incubation period, which should not exceed 4hr. This incubation period is sufficiently long to determine the metabolic stability and to allow identification of the main metabolites of a test substance, but may be too short to allow generation of some minor, particularly phase II metabolites, that contribute less than 3% to total metabolism. To achieve longer incubation periods, hepatocyte culture systems or bioreactors are used. In this research program, two bioreactor systems have been optimized: the perifusion culture system and 96-well plate bioreactors. The perifusion culture system consists of collagen-coated slides allowing the continuous superfusion of a hepatocyte monolayer with culture medium as well as establishment of a constant atmosphere of 13% oxygen, 82% nitrogen, and 5% CO2. This system is stable for at least 2 weeks and guarantees a remarkable sensitivity to enzyme induction, even if weak inducers are tested. A particular advantage of this systemis that the same bioreactor can be perfused with different concentrations of a test substance in a sequential manner. The 96-well plate bioreactor runs 96 modules in parallel for pharmacokinetic testing under aerobic culture conditions. This system combines the advantages of a three-dimensional culture system in collagen gel, controlled oxygen supply, and constant culture medium conditions, with the possibility of high throughput and automatization. A newly developed co-culture system of hepatocytes with intestinal bacteria offers the possibility to study the metabolic interaction between liver and intestinal microflora. It consists of two chambers separated by a permeable polycarbonate membrane, where hepatocytes are cultured under aerobic and intestinal bacteria in anaerobic conditions. Test substances are added to the aerobic side to allow their initial metabolism by the hepatocytes, followed by the metabolism by intestinal bacteria at the anaerobic side. Precision-cut slices represent an alternative to isolated hepatocytes and have been used fo the investigation of hepatic metabolism, hepatotoxicity...

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The objective of the present study is to visualize differences in the body distribution between radiolabelled AZT bound to nanoparticles and a control solution. Polyhexylcyanoacrylate nanoparticles were manufactured by emulsion polymerization in the presence of AZT and an ionic emulsifier, bis(2-ethylhexyl) sulfosuccinate sodium. The AZT-control solution was equally prepared, but contained no monomer. The two preparations were administered either by i.v. injection or perorally by gavage. After determined time points the animals were sacrificed using carbon dioxide. The cadavers were shock-frozen in cellulose gel and cut into slices using a cryomicrotome. The tissue cross sections were fixed on an adhesive tape and then were freeze dried. The quantification of the radioactive AZT in the different organs and tissues was performed by radioluminography, and the images were generated on a computer. After i.v. injection of AZT-nanoparticles, a high amount of the AZT label was found in the organs belonging to the reticuloendothelial system. In these organs the radioactivity was inhomogeneously distributed showing that the uptake of the particle-associated radioactivity depended on the type of the cells located in the organs and was consistent with uptake by macrophages. The highest radioactivities were found in the GI-tract and in the liver. A difference in the elimination pathway between AZT-control solution and AZT bound to nanoparticles also was visible on the images. Similar results were obtained after oral administration. Of course, with the latter route a larger portion of AZT remained in the GI-tract especially after administration of nanoparticle-bound drug. These results confirmed those obtained by a classically performed quantitative whole body distribution study using liquid scintillation. This demonstrates that radioluminography is a useful method to study the organ distribution of drugs bound to nanoparticles.

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