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Browsing by Subject "maksatoksisuus"

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  • Porola, Pauliina (2012)
    Hepatotoxicity is an undesired feature of many drugs and is one of the main reasons for attrition during the drug development process. Although an in vitro model can never totally correspond to or replace a whole organism, a reliable in vitro model for liver toxicity screening would help to detect liver toxicity earlier in the development process. Effective and early in vitro screening would reduce the need of animal subjects and clinical trials and thus would be both ethically more acceptable and more cost-effective. Currently mostly used models for liver metabolism and toxicity studies are primary hepatocytes, hepatic cell lines and animal models. However, these models have many drawbacks and are not considered reliable. Human embryonic stem cells (hESCs) are pluripotent cells that can be differentiated into many specialized cell types including hepatocytes. They are also self-renewable and thus represent an unlimited and promising source of hepatocytes to be used as a tool in in vitro liver toxicity testing of drug candidates. The aim of this study was to produce hepatocytes from hESCs via multiple steps following the in vivo pathway of developing hepatocytes: first hESCs were differentiated into definitive endoderm cells, after which they were differentiated into hepatic progenitor cells. Finally, hepatocyte-like cells (HLCs) were induced from the progenitor cells. Our specific interest was the use of hepatic cell derived acellular matrix as a differentiation basis for hepatic progenitors and hepatocytes. We also studied the effect of Matrigel overlay on the hepatic differentiation. Differentiation method without the Matrigel overlay was promising. HLCs showed correct hepatocyte-like morphology and expressed hepatocyte markers such as albumin, α-antitrypsin, CYP3A4 and HNF4α both on mRNA and protein level shown by qPCR and flow cytometry and immunofluorescence staining, respectively. Accordingly, the expression of stem cells marker SSEA-3 showed a tendency to decrease as the differentiation proceeded. HLCs also functionally resembled hepatocytes shown by albumin production. However, we could not detect other hepatocyte functions such as urea production or CYP activity. With Matrigel overlay, the hepatocyte-like morphology of the cells was lost, no albumin production was shown and the expression of several hepatocyte markers was lower than in the experiment done without the Matrigel overlay. Thus, Matrigel overlay was shown to be unbeneficial for hepatocyte differentiation. In conclusion, we showed that differentiation of hESCs on the acellular matrix with specific growth factors and without the Matrigel overlay seems promising as a method to produce HLCs. This preliminary study serves as a basis for future studies, in which the differentiation method should still be further studied and developed to yield functional HLCs of uniform quality.
  • Mustonen, Merja (2011)
    Drug induced liver injury is one of the frequent reasons for the drug removal from the market. During the recent years there has been a pressure to develop more cost efficient, faster and easier ways to investigate drug-induced toxicity in order to recognize hepatotoxic drugs in the earlier phases of drug development. High Content Screening (HCS) instrument is an automated microscope equipped with image analysis software. It makes the image analysis faster and decreases the risk for an error caused by a person by analyzing the images always in the same way. Because the amount of drug and time needed in the analysis are smaller and multiple parameters can be analyzed from the same cells, the method should be more sensitive, effective and cheaper than the conventional assays in cytotoxicity testing. Liver cells are rich in mitochondria and many drugs target their toxicity to hepatocyte mitochondria. Mitochondria produce the majority of the ATP in the cell through oxidative phosphorylation. They maintain biochemical homeostasis in the cell and participate in cell death. Mitochondria is divided into two compartments by inner and outer mitochondrial membranes. The oxidative phosphorylation happens in the inner mitochondrial membrane. A part of the respiratory chain, a protein called cytochrome c, activates caspase cascades when released. This leads to apoptosis. The aim of this study was to implement, optimize and compare mitochondrial toxicity HCS assays in live cells and fixed cells in two cellular models: human HepG2 hepatoma cell line and rat primary hepatocytes. Three different hepato- and mitochondriatoxic drugs (staurosporine, rotenone and tolcapone) were used. Cells were treated with the drugs, incubated with the fluorescent probes and then the images were analyzed using Cellomics ArrayScan VTI reader. Finally the results obtained after optimizing methods were compared to each other and to the results of the conventional cytotoxicity assays, ATP and LDH measurements. After optimization the live cell method and rat primary hepatocytes were selected to be used in the experiments. Staurosporine was the most toxic of the three drugs and caused most damage to the cells most quickly. Rotenone was not that toxic, but the results were more reproducible and thus it would serve as a good positive control in the screening. Tolcapone was the least toxic. So far the conventional analysis of cytotoxicity worked better than the HCS methods. More optimization needs to be done to get the HCS method more sensitive. This was not possible in this study due to time limit.