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in the gut) that were available in the literature<br />
were also provided.<br />
Level 4 of the tiered approach was reached<br />
only in a limited set of models. Permeability<br />
in in vitro systems was not easily<br />
connected with the Fraction absorbed<br />
measured in clinical studies. On the other<br />
hand, HepaRG resulted in prediction very<br />
close to that of pooled human primary<br />
hepatocytes. Moreover, the quality of prediction<br />
was as good as that obtained with<br />
hepatocytes. This suggest that HepaRG<br />
can represent a valid surrogate of pooled<br />
human primary hepatocytes, with the advantage<br />
to be more consistent and reproducible.<br />
Conclusions<br />
An optimised in vitro model of hepatocytes<br />
is now available, well characterised for<br />
different functions and for the response<br />
to specific toxicants: the HepaRG cell<br />
line. The project has also supported new<br />
lines of research for obtaining functional<br />
hepatocytes in vitro, in order to promote<br />
the availability of more than one in vitro<br />
model, thus amplifying the opportunities.<br />
The transfection approach with multiple<br />
factors inducing the expression of major<br />
metabolic activities has been successfully<br />
accomplished; however the procedure only<br />
succeeded in Hela cells but not in hepatic<br />
cell lines. The TSA treatment to induce<br />
hepatic differentiation has proved effective<br />
in human mesenchymal progenitor cells,<br />
for which isolation protocols have also<br />
been set up.<br />
The intestinal model that has shown<br />
to be the most similar to normal small<br />
intestinal enterocytes in vivo was the<br />
Caco-2 cell line. These cells have been<br />
characterised at genomic and functional<br />
levels. Differentiated Caco-2 cells<br />
seem the model of choice for oral drug<br />
absorption, especially for active uptake,<br />
less for secretion. An optimised method<br />
of maintenance of the cells has been set<br />
up, which allows a strict control of the<br />
differentiation process. This approach<br />
has shown that source of the cells or the<br />
number of passages (e.g., sub-cultures)<br />
are not so critical if the culture procedure<br />
is strictly followed. Other aspects have<br />
been investigated in relation to the optimal<br />
expression of the phenotypic and genotypic<br />
markers: cell differentiation on permeable<br />
substrate (i.e., filters) is the only one that<br />
guarantees the correct polarisation and<br />
differentiation of the cells. Moreover, since<br />
the serum is still a factor of variability and<br />
interference especially in toxicity testing,<br />
synthetic media have successfully been<br />
tested and it has also been also shown<br />
that serum addition is not needed in the<br />
apical medium.<br />
Concerning transport and metabolic<br />
activities, while they have been well<br />
characterised in the cellular models<br />
used in the project, it has not been<br />
possible to assess them with respect to<br />
a wider choice of compounds, since the<br />
extensive characterisation of the models<br />
has requested more time and work than<br />
expected. That has been also the reason<br />
for the request for six-month extension of<br />
the project.<br />
This situation has also had an impact<br />
on the in silico modelling work, as only a<br />
PROGRESS REPORTS FROM EU-FUNDED PROJECTS<br />
Progress Report 2011 & AXLR8-2 Workshop Report<br />
113
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