Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Signal transduction wikipedia , lookup
Cytokinesis wikipedia , lookup
Extracellular matrix wikipedia , lookup
Cell growth wikipedia , lookup
Cell encapsulation wikipedia , lookup
Cellular differentiation wikipedia , lookup
Tissue engineering wikipedia , lookup
Cell culture wikipedia , lookup
AltexSupl-087-100 01.08.2007 13:40 Uhr Seite 93 PROJECTS IN PROGRESS these cultures will be assessed by inoculation of 2D (microslides) HCT-8 cells which are analysed by qPCR. Fig. 1: HCT-8/CCL6 cells growing on Cytodex 3 microcarriers in Rotary Cell Culture System vessels. A) after 4 days; B) after 21 days of cultivation. membrane and extracellular matrix components (laminin, fibronectin, collagen IV) as described. Mucus production is demonstrated by histochemical staining with PAS and by using FITC-labelled lectins. 3D cell aggregates are used for short-term infection assays with oocysts of Cryptosporidium in 24-well tissue culture plates. Early events of attachment and internalisation of sporozoites will be monitored by using differently labelled (fluorescein, Cy3) monoclonal antibodies directed against Cryptosporidium, with and without cell permeabilisation. Intracellular growth of the parasite will be analysed by transmission electron microscopy in time course studies. Analysis of apoptosis/necrosis following inoculation with Cryptosporidia in 3D aggregates will be done after dissociation into individual cells by treatment with 0.1% EDTA and incubation with FITCannexin V and propidium iodide and compared to non-infected cells. In addition, 3D cell aggregates will be used for long-term propagation of Cryptosporidium directly in the RCCS vessels (55 ml), and the 3D aggregates will be analysed for intracellular parasite growth by fluorescence microscopy using FITClabelled mAb, by transmission electron microscopy and by quantitative PCR (qPCR). The viability of oocysts produced in Conclusions and relevance for 3R This start-up project should yield a robust protocol for growing 3D aggregates of intestinal epithelial cells which represent a growth environment similar to that between brush border microvilli of epithelial cells and therefore might allow to propagate previously uncultivable organisms and might serve as a relevant model for a wide variety of pathogens (virus, bacteria, parasites) of medical and veterinary importance which infect epithelia of the gastrointestinal, but also the respiratory or urogenital tract. Therefore, our work will contribute to bridge the gap between experiments done with 2D conventional cell cultures with their inherent limitations and those performed in live animals. By providing a better model for what happens in vivo, these 3D cultures will allow researchers to considerably reduce their use of experimental animals in their investigations on a variety of infectious agents. References Nickerson, C. A., Goodwin, T. J., Terlonge, J. et al. (2001). Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serovar Typhimurium pathogenesis. Infect. Immun. 69, 7106. Correspondence to PD Dr. Alexander Mathis Institute of Parasitology University of Zurich 8057 Zurich Switzerland e-mail: [email protected] Establishment of a Murine Syngeneic Co-Culture System of Intestinal Epithelial Cells with Intraepithelial T Lymphocyte Subsets Christoph Mueller Institute of Pathology, University of Bern, Bern, Switzerland Keywords: human, mice, endothelia, T cell, molecular biology, PCR, reduction, replacement Background and aim Investigations of the mutual cellular interactions between epithelial cells and intraepithelial lymphocytes (IEL) in the intestine are difficult to achieve due to the limited accessibility www.forschung3r.ch/en/projects/pr_98_05.html ALTEX 24, Special Issue 2007 of this compartment to direct experimental interventions. Furthermore, relevant functional parameters, such as the influence of distinct T cell subsets on barrier function of the intestinal epithelium are difficult to determine in experimental animals. These aspects highlight the importance of establishing in vitro systems to obtain relevant information on the inter93 AltexSupl-087-100 01.08.2007 13:40 Uhr Seite 94 PROJECTS IN PROGRESS Tab. 1: Representative example of a comparative isolation of human intraepithelial lymphocytes by Percoll density gradient: Equal numbers of cells, released from the intestinal epithelium, were subjected to either conventional Percoll gradient, or elutriation centrifugation, respectively. Percoll (44/67%) Fig. 1: Scatter blot of the IEL-enriched fraction following elutriation centrifugation. In this preparation 88% of the cells are intraepithelial lymphocytes. play of these cellular systems in maintaining local tissue homeostasis. The establishment of suitable in vitro co-culture systems was so far hampered by the poor yield of IEL isolated from intestinal epithelium and particularly the short survival of some IEL subsets which may be < 12 hours in vitro under normal T cell culture conditions. Hence, the specific aims of the present proposal are 1. to increase the yield of IEL isolated from the intestinal epithelium of mice and humans 2. to optimise culture systems for extended culture periods for all IEL subsets 3. to establish co-cultures of these IEL subsets with syngeneic intestinal epithelial cell lines and to determine whether and how antigen-specific activation of IEL affects the permeability of the intestinal epithelial cell layer and the pro- and anti-inflammatory properties of epithelial cells. Method and results Protocols for the reproducible separation of intraepithelial lymphocytes (IEL) from contaminating epithelial cells by elutriation centrifugation will be established. With this technique we have already managed to substantially enhance the yield of IEL from human intestinal tissue samples (Tab. 1) and to obtain a highly enriched fraction of IEL (Fig. 1). Currently, we are also optimising this method for the isolation of mouse IEL for subsequent co-culture with a syngeneic (H-2b) intestinal epithelial cell line (mICCl2, YAMC or MSIE). Culture conditions in vitro need to be established and optimised to allow for an extended co-culture of all IEL subsets with these epithelial cell lines. The mutual interactions and consequences of an IEL-epithelial cell culture will be monitored using transepithelial resistance measurements, gene expression profiles of all cell populations involved (e.g. using Realtime RT-PCR procedures), and ELISA- and Luminex®-based methods to determine secretion of cytokines. 94 Elutriation Centrifugation 7 8 x 10 7 9 x 10 (11%) 6 9 x 10 (11%) 6 % T cells in IEL enriched fraction 25% 61% Yield of CD3 T cells (in %) 2.3 x10 (26%) Total number of cells (IEC and IEL) after EDTA/DTT incubation („starting material“) 8 x 10 Total number of human small intestinal IELin starting material 6 8.0 x 10 (89 %) 6 Conclusions and relevance for 3R Reduction in animal experimentation: The establishment of the envisaged syngeneic intestinal epithelial cell – intraepithelial lymphocyte co-culture system should allow to directly monitor time – dependent changes in these cell populations. Furthermore, it will allow direct experimental interventions such as antigen-specific activation of the IEL subsets (using MHC-restricted peptides), and mimicking an inflammatory situation by adding either recombinant pro-inflammatory cytokines or relevant inflammatory cells. Refining experimental methods involving live animals: The optimisation of the isolation protocols for intestinal IEL will not only lead to a more representative and thus more reproducible population of IEL, but may also substantially reduce the number of donor animals required for the isolation of IEL. Furthermore, the epithelial cell lines can be genetically modified (e.g. using siRNA) to experimentally assess the direct functions of candidate genes and their products relevant for the IELepithelial cell interactions. In the future, this may make the generation and the use of genetically modified mice often obsolete for studying intestinal IEL functions and lymphocyte-epithelial cell interactions. Last but not least, with these co-culture systems we expect to obtain relevant information regarding the biology of intestinal IEL that will allow more precise analysis and interpretation of the pathophysiological role of IEL in humans. Correspondence to Prof. Dr. Christoph Mueller Institute of Pathology University of Bern 3010 Bern Switzerland e-mail: [email protected] ALTEX 24, Special Issue 2007