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Table 1. Values are expressed as percent of control cells differentiated in medium with 10%
FBS in apical and basal compartments (control). Mean of 3-4 experiments performed in
triplicate.
Parameters Measured at Day
21 from Seeding
10% FBS in Basal
Medium
% of control MITO+ serum extender
in Basal medium
% of control
Permeability (mannitol 99.24 177.88
passage)
PGP activity (BL to AP digoxin) 144.35 158.23
PEPT1 activity (cephalexin AP 105.75 202.39
to BL transport)
Alkaline phosphatase activity 90.19 62.43
Sucrase activity 104.41 41.24
the cell border, compared to cells differentiated
on plastic or glass.
INRAN also tried a chemically defined
culture medium without fetal bovine serum
(FBS) supplementation to reduce the
variability introduced by the variable and
unknown composition of serum batches
employed for cell culture media. The replacement
of FBS from the culture medium
with a chemically defined supplement
(MITO+ serum extender) allowed a good
degree of differentiation of the cell line
under better controllable and reproducible
conditions. In particular, optimal development
of barrier properties (mannitol passage)
and expression of active transport
activities (PGP and PEPT1) was obtained
in chemically defined medium. Lower enzyme
activities (alkaline phosphatse and
sucrase) were detected in serum-free medium.
In addition, it was shown that even
when FBS was used in the culture medium,
its presence in the apical medium was
not necessary to allow optimal functional
differentiation, as shown by all parameters
investigated (Table 1). Unfortunately, the
exact composition of the MITO+ serum
extender is not released by the manufacturer,
thus reducing its potential as an optimal
‘chemically defined’ medium, as its
reproducibility rests upon its production
quality. It can therefore be concluded that
FBS can be replaced by MITO+ serum extender
in the basal medium during differentiation
of Caco-2 and in any case, even
if FBS is used, it should be omitted from
the apical medium to reduce its costly and
unethical use.
Transport Activities
Since the aim of the project was to obtain
a well-characterised cell line representative
of human small intestinal enterocytes,
expressing absorptive and metabolic functions,
another set of studies dealt with the
characterisation of the transport activi-
104 PROGRESS REPORTS FROM EU-FUNDED PROJECTS
Progress Report 2011 & AXLR8-2 Workshop Report