In vitro system for the prediction of hepatotoxic effects in primary hepatocytes.

Prediction of liver toxicity and compound responses continues to be a major challenge for the pharmaceutical industry. In vitro studies on liver cells have been developed to reduce or replace animal experiments. However, most of the tests in use are based on cell lines which do not necessarily represent normal cell physiology. We compared the response of primary human hepatocytes from two donors with primary rat hepatocytes and the cell line HepG2 to the test compound acetaminophen (AAP) by measuring oxygen consumption, extracellular acidification and cell adhesion as dynamic parameters of cell metabolism. Primary human hepatocytes were cultured on collagen pre-coated sensor chips or in conventional two-dimensional cultures in chemically defined Human Hepatocyte Maintenance Medium. This medium allows cultivation of functionally differentiated hepatocytes for several weeks. Sensor chip based results were compared with conventional assays for hepatocytes like albumin release and urea release. The hepatocytes were exposed to AAP (50-2815 mg/l) for 24 h. Cell respiration was inhibited by AAP concentrations of 500 mg/l and more in all three cell types, whereas only the cellular acidification rates and cell adhesion of the rat hepatocytes and the HepG2 cells were affected by AAP. In conventional cultures of human hepatocytes, AAP had no effect on cellular viability. Whereas high doses of AAP (2815 mg/l) diminished albumin secretion by 70-80%.

Online monitoring of cell metabolism for studying pharmacodynamic effects.

To characterize modes of action of substances and their cytotoxic effects Bionas GmbH has developed a new screening system to allow the continuous recording of how an active substance can act (Bionas 2500 analyzing system). In the pharmaceutical industry it is important to acquire as much information as possible about the metabolic effects of an active substance. Most classical pre-clinical studies are very expensive and time-consuming. Often they are so-called end-point tests which require many individual tests before approximate statements can be made about how an effect takes its course. With the Bionas 2500 analyzing system metabolically relevant data including oxygen consumption, acidification rate and the adhesion (cell impedance) of cells can be measured in parallel, online and label-free. Using e.g. ion-sensitive field effect-transistors (ISFET) and electrode structures it is possible to observe metabolic parameters non-invasively and continuously over longer periods of time. The system has already been established for several cell models, cell lines as well as primary cells. It also offers the advantage that regenerative effects can be observed during the same test run.

A new method to assess drug sensitivity on breast tumor acute slices preparation.

A method for assessing tumor drug sensitivity is described that is based on preparation of tissue slices and use of silicon chips equipped with electrochemical sensors (multisensor array). The tumor slices (200-300 microM thick) are prepared after surgery and incubated in a medium for recovery after slicing. The advantage, compared to other preparations, is that the original three-dimensional structure is retained. Multisensor arrays measure: (a) pericellular acidification (anaerobic metabolism) and (b) oxygen consumption (respiration). The innovative aspect is that such measurements can be made online, as opposed to using a large battery of endpoint tests on cell vitality and proliferation. Electron microscopy of slices serves to determine cell density and structure and induction of apoptosis/necrosis. Slices of more than 200 breast tumors were used. Metabolic activity was inhibited by sodium fluoride, which reduces glycolysis, and potassium cyanide, which inhibits respiration. These changes are thus reflected in the curves of acidification and oxygen consumption. In other experiments the cytostatic Taxol, an anticytoskeletal agent, was used showing dose and time-dependent effects on acidification and oxygen consumption. In conclusion, the method presented here, is able to provide information on drug sensitivity of a tumor, which aids in designing individualized therapy and is used for drug screening.