Microfluidic chip for monitoring Ca
2+
transport
through a confluent layer of intestinal cells†
C. Huang,‡
*
a
Q. Ramadan,§
*
a
J. B. Wacker,{
a
H. C. Tekin,k
a
C. Ruffert,**
a
G. Verg
`
eres,
b
P. Silacci
b
and M. A. M. Gijs
a
The absorption of dietary calcium through the intestinal barrier is
essential for maintaining health in general and especially of the bone
system. We propose a microfluidic model that studies free calcium
(Ca
2+
) transport through a confluent monolayer of Caco-2 cells. The
latter were cultured on a porous membrane that was positioned in
between a top and bottom microfluidic chamber. Fresh cell culture
medium was continuously supplied into the device at a flow rate of 5
nL s
1
and the culture progress of the cell monolayer was continu-
ously monitored using integrated Transepithelial Electrical Resistance
(TEER) electrodes. The electrical measurements showed that the
Caco-2 monolayer formed a dense and tight barrier in 5 days. The
transported free Ca
2+
from the top microfluidic chamber to the
basolateral side of the cell monolayer was measured using the
calcium-sensitive dye fura-2. This is a ratiometric dye which exhibits
an excitation spectrum shift from 340 nm to 380 nm, when it binds to
Ca
2+
with an emission peak at 510 nm. Therefore, the concentration of
free Ca
2+
is proportional to the ratio of fluorescence emissions
obtained by exciting at 340 nm and 380 nm. The barrier function of the
cell monolayer was evaluated by a measured rate of Ca
2+
transport
through the monolayer that was 5 times lower than that through the
bare porous membrane. The continuous perfusion of cell nutrients
and the resultant mechanical shear on the cell surface due to the fluid
flow are two key factors that would narrow the gap between the in
vivo and in vitro conditions. These conditions significantly enhance the
Caco-2 cell culture model for studying nutrients bioavailability.
Introduction
Dietary calcium intake has an important impact on human
health and chronic calcium deciency resulting from inade-
quate intake or poor intestinal absorption is a major cause of
reduced bone mass and osteoporosis. Calcium is absorbed in
the mammal along two routes: (i) a transcellular mechanism,
predominant in the duodenum and regulated by vitamin D, and
(ii) a paracellular, concentration-dependent diffusional process
that takes place throughout the length of the intestine.
1
In
normal conditions, calcium predominantly traverses the intes-
tinal epithelium via the paracellular pathway,
2
which is driven
by the voltage gradient between the apical and basolateral side
of the gut epithelium, and regulated by the tight junction
proteins, which polymerize to form an array of channel-like
paracellular pores.
3
In vivo methods, which provide direct data
of bioavailability, have been used to measure the bioavailability
of a great variety of metabolites.
4,5
Such studies imply the
consumption of a certain dose of a nutrient by either humans or
animals, and subsequently monitoring its concentration in the
blood plasma over time and compare it to an equivalent
nutrient dose found in a food source.
6
However, in vivo studies
are technically difficult, costly, and limited by ethical
constraints. Another major drawback of in vivo data is the
variability in physiological state of individuals and the possible
interaction of the nutrient with other components in the diet.
7
Therefore, alternative methods are needed.
In vitro models of the human gastrointestinal tract (GIT),
that closely mimic the physiological processes of absorption,
may provide efficient tools for the bioavailability measure-
ments, and therefore can show more systematic human tissue
response to nutrients and signicantly reduce experimentation
expenses. The standard in vitro model used for studying the
bioavailability of nutrients in food is a conuent layer of
epithelial cells,
8,9
with Caco-2 cells the most popular choice for
modeling the human GIT.
10,11
In this model, the cell monolayer
is grown on a porous membrane, such as a Transwell insert
(Millipore, USA), which is placed inside a culture well to form a
a
Laboratory of Microsystems,
´
Ecole Polytechnique F´ ed´ erale de Lausanne, Switzerland.
E-mail: alramadanq@ime.a-star.edu.sg
b
Institute for Food Science, Agroscope, Federal Office of Agriculture, Berne, Switzerland
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra09370d
‡ College of Chemical Engineering, Nanjing Forestry University (NFU), Nanjing,
210037, P. R. China.
§ Bioelectronics Department, Institute of Microelectronics, A*STAR, Singapore.
{ CSEM, Rue Jaquet-Droz 1, CH-2002 Neuchˆ atel, Switzerland.
k Stanford University School of Medicine, Canary Center Early Cancer Detection,
Stanford, CA 94305-5101, USA.
** Institut fuer Mikroproduktionstechnik, Produktionstechnisches Zentrum,
Leibniz Universitaet Hannover, 30823 Garbsen, Germany.
Cite this: RSC Adv. , 2014, 4, 52887
Received 28th August 2014
Accepted 16th October 2014
DOI: 10.1039/c4ra09370d
www.rsc.org/advances
This journal is © The Royal Society of Chemistry 2014 RSC Adv. , 2014, 4, 52887–52891 | 52887
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