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Precision cut tissue slices (PCTS) represent a suitable and convenient tool for pharmacological, toxicological and morphological studies. Cryopreservation would enable to overcome the shortage of liver tissue, in particular in settings using human liver tissue. We investigated the potential of cryopreservation of porcine PCTS as a morphological tool by rapid freezing with 10% and 30% dimethyl sulfoxide as cryopreservation agents and with or without medium using a Brendel/Vitron tissue slicer. Incubation after thawing was done in a static incubation system. Slices were cultured for 3 h, 6 h, 24 h and 48 h and assessed histologically and immunohistologically for proliferation (Ki67) and spontaneous as well as induced apoptotic activity (M30Cytodeath). Vitality was tested using the Tox-8 test. After cryopreservation, morphology of PCTS was well preserved up to 24 h. A reduction of vitality rate took place. Compared to non-frozen PCTS, the rate of spontaneous proliferation of Kupffer cells and apoptosis of hepatocytes were significantly reduced independent of the freezing conditions. The reactivity of PCTS to apoptotic stimuli was significantly reduced in tissue slices after cryopreservation. Apoptotic stimuli could not induce the same amount of cell deaths compared to non-frozen sections. Thus, cryopreservation of PCTS does interfere with pathomechanisms of apoptosis in PCTS., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Background: Therapeutic options in patients with metastatic colorectal cancer are still limited. As apoptosis contributes to the overall sensitivity to radiotherapy or chemotherapy, a better understanding of the apoptotic process in metastatic tumour tissues is necessary., Materials and Methods: Precision cut tissue slices (PCTS) of three human liver metastases were used to investigate the effect of activating CD95 antibodies (concentrations: 0.1 μg/ml, 1 μg/ml and 1 μg/ml and 1 μg/ml actinomycin D) as well as TNFα (concentrations 1 ng/ml; 10 ng/ml and 10 ng/ml and 1 μg/ml actinomycin D) directly in tumour tissue after 6 h, 12 h and 24 h. The apoptotic effect was assessed immunohistochemically., Results: Activating CD95 antibodies combined with actino-mycin D led to a significant increase in apoptosis after 12 h. Using TNFα at a high dosage, a significant increase in the apoptosis rate was observed after 6 h and after 12 h in all dosage groups., Conclusions: PCTS can be used to investigate the effect of different apoptotic signals directly in human tumour tissues. TNFα is able to effectively induce apoptosis in liver metastases of colorectal carcinoma. Thus, the extrinsic pathway of apoptosis may be a promising target in the development of new therapeutic approaches.

Nonsteroidal anti-inflammatory drugs are known to reduce the risk and mortality from colorectal carcinoma by inhibiting cyclo-oxygenases (COX). COX-2 expression was investigated immunohistologically in 57 patients with colorectal carcinomas and in the corresponding liver metastases using tissue microarray analysis. Ex vivo COX-2 inhibition with assessment of apoptosis was performed using precision-cut tissue slices of three human liver metastases. Following stimulation with different concentrations of the selective COX-2 inhibitor meloxicam, apoptosis was assessed immunohistochemically after 6 h and 12 h. All primary carcinomas and 56 out of the 57 liver metastases showed various degrees of cytoplasmatic COX-2 expression being with a reduction and in the liver metastases. There was a time- and concentration-dependent change in the number of apoptotic cells in tissue slices, however, this was without statistical significance. COX-2 is constantly involved in the carcinogenesis and metastatic process of colorectal cancer. The antineoplastic effect of COX-2 inhibition may be based on different pathways, including changes in sensitivity to apoptosis.

Unlabelled: The conversion of epithelial cells in a mesenchymal cell type is called "epithelial-mesenchymal-transition" (EMT). This process is defined by a loss of epithelial specific characteristics such as cell adhesion, polarity and a reorganization of cytoskeletal proteins. EMT has been shown to be involved in progression of cancer and in obstructive renal fibrosis. In this study we analyzed liver tissues in a bile-duct ligation model of rats and human liver biopsies with cholestatic fibrosis and chronic hepatitis c infection to determine if biliary epithelial cells undergo phenotypical and functional changes during chronic injury., Methods: Liver tissue of rats and human patients was examined by immunohistochemistry using antibodies against epithelial and mesenchymal specific targets as well as molecules of potentially activated signaling pathways. To study contribution of biliary epithelial cells in extracellular matrix production we performed laser microdissection combined with real-time PCR., Results: Bile duct ligation in rats induced a prominent biliary epithelial proliferation and a pronounced expression of vimentin was observed in biliary epithelial cells, whereas no vimentin expression was detectable in bile duct cells of sham operated rats. In human liver biopsies from patients with cholestatic fibrosis and chronic hepatitis c infection a prominent biliary expression of vimentin could be shown. Despite this, epithelial marker proteins were still detectable. Further, we observed collagen I mRNA expression in laser microdissected bile ducts., Conclusion: Biliary epithelial cells show cytoskeletal rearrangements during chronic liver injury towards a mesenchymal phenotype. The detection of collagen I mRNA in bile duct cells suggests that they might participate in extracellular matrix production.

Cultures of precision-cut tissue slices allow the investigation of substance effects on human tissues under in vivo-like conditions over a limited time span. We have adapted the model for direct analyses of antineoplastic substances on tumor tissues. We have recently demonstrated that selective cyclooxygenase-2 (COX-2) inhibitors strongly suppress growth of human hepatocellular carcinoma (HCC) cells in vitro and nude mouse HCC implants by inducing apoptosis and reducing proliferation. We have now analyzed the effects of COX-2 inhibition on human tumor tissue. Three hundred micrometer slices of tumorous and non-tumorous liver tissue from three surgically resected HCCs were cultured with increasing concentrations of the selective COX-2 inhibitor Meloxicam (20-200 microM) for 6, 12, 24, and 48 h. The cultured tissue slices were analysed morphologically and by immunohistology for proliferation (Ki-67), apoptosis (M30), and COX-2 expression. COX-2 was expressed in all HCCs and in the non-tumorous liver tissue. Cytoplasmic COX-2 immunoreactivity in HCCs increased during culturing time. In two of three cases, COX-2 inhibition significantly increased tumor cell apoptosis in HCCs, whereas the low basal apoptosis rate in the non-tumorous liver parenchyma did not change. Tumor cell proliferation was mildly reduced, but the changes did not reach statistical significance. These results demonstrate that the precision-cut tissue slice culture model is a useful tool to analyze directly drug-dependent antitumorous or unwanted organ-specific effects. The analysis of COX-2 inhibition lends further support to the antineoplastic effects previously demonstrated in vitro and in animal models.