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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