MICROFLUIDIC DEVICE TO STUDY THE INTERPLAY OF LIVER AND
INTESTINE IN THE REGULATION OF BILE ACID SYNTHESIS
Paul M. van Midwoud1,2, Marjolijn T. Merema1, Elisabeth Verpoorte2, Geny M. M. Groothuis1
1
Pharmacokinetics, Toxicology and Targeting and 2Pharmaceutical Analysis, University of Groningen,
THE NETHERLANDS
ABSTRACT
Recently, we introduced a microfluidic biochip to incorporate intestinal and liver slices and demonstrated its proper
functioning by comparing the metabolic capacity of slices in the biochip with those in well plates [1, 2]. We now show that
interorgan interactions can successfully be studied by sequential perifusion of intestinal and liver slices in the biochip by
investigating the role of the intestine in bile salt-induced regulation of Cytochrome P450 7A1 (CYP7A1) in the liver.
KEYWORDS: Liver slices, Intestinal slices, Interorgan interaction, Cytochrome P450 7A1, Fibroblast growth factor 15
(FGF15)
INTRODUCTION
The synthesis of bile acids mainly occurs in the liver, with CYP7A1 as the key enzyme. The expression of this enzyme
in the liver is regulated by several mechanisms. Bile salts play a prominent role in the activation of the farnesoid X receptor
(FXR)-mediated feedback down-regulation of CYP7A1 in the liver. In addition, the intestine plays an important role in the
regulation of CYP7A1 [3]. Primary bile acids such as chenodeoxycholic acid (CDCA) induce the expression of fibroblast
growth factor 15 (FGF15 in rodents or FGF19 in human) in the intestine. In vivo FGF15 is transported via the portal vein
to the liver, where it causes a down-regulation of CYP7A1. We investigated whether this second pathway could be
confirmed in vitro with intact precision-cut tissue slices of intestine and liver.
We recently developed a new system for the incubation of rat liver slices, consisting of a perfused microfluidic biochip
where the liver slice is continuously perifused. (note: the term “perifusion” is used to emphasize that the medium flows
around the tissue slice rather than through it, whereas the term “perfusion” applies to the medium flow through the microchamber.). The perfused biochip contains two polycarbonate membranes (10 µm thick) which form the ceiling and floor
of each microchamber to realize an even distribution of medium flow around the tissue slice and to ensure that the slice is
horizontally suspended in the flow (Figure 1). The chip also contains two 250-µm-thick PDMS membranes above and
below each chamber to act as acting as “breathing” membranes to keep the pH and oxygen levels stable in the incubation
environment.
PDMS membrane
(250 µm)
Outlet
Polycarbonate membrane
(10 µm, 8 µm pores)
Tissue slice
Polycarbonate membrane
(10 µm, 8 µm pores)
PDMS membrane
(250 µm)
Inlet
Figure 1. Cross-sectional view of one chamber with the integrated polycarbonate (10 µm thick) and PDMS membranes (250
µm thick). The microchamber is Ø 4 mm x 2 mm high (volume of 25 µL).
The investigation of interorgan effects can be achieved by coupling two microchambers, one containing an intestinal
tissue slice, the other a liver slice, which can be sequentially perfused. In this way, metabolites formed by the intestine in
the first chamber will be directed to the liver in the second chamber, thereby mimicking in vivo, first-pass metabolism. By
sequential perifusion of intestinal and liver slices, the interplay of the two organs in the regulation of bile acid synthesis will
be studied.
EXPERIMENTAL
Single liver and ileum slices were perifused for 7 hours with medium with or without 50 µM CDCA in the biochip
shown in Figure 1. For sequential perfusion, the outlet of the chamber with the ileum slices was connected to the inlet of
the second chamber with the liver slice (Figure 2). The mRNA expression of FGF15 in the ileum slices was measured by
RT-PCR with villin as housekeeping gene. In liver slices, the expression of CYP7A1 was measured with GAPDH as housekeeping gene. The gene expression in the CDCA-treated slices was normalized to that in the slices incubated for 7 hours
without CDCA. This study was performed in three rats with three slices per experiment (rat).
978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS
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14th International Conference on
Miniaturized Systems for Chemistry and Life Sciences
3 - 7 October 2010, Groningen, The Netherlands
5% CO2 / 95% O2
Reservoir
Liver slice
Polycarbonate
membrane
“Breathing” membrane
Intestinal slice
Polycarbonate
membrane
“Breathing” membrane
Microdevice
Syringe pump (10 µL/min)
Figure 2. Schematic view of the set-up for sequential perifusion of intestinal and liver precision-cut slices.
RESULTS AND DISCUSSION
When intestinal slices were treated with CDCA, FGF15 was highly upregulated in the intestinal slices from all three rats.
As expected, no significant differences were obtained between the intestinal slices perfused in a single biochip (166 fold)
and those perfused sequentially with liver slices (151 fold). The expression of CYP7A1 in the liver slices was not affected
by sequential perfusion without CDCA. In contrast, when CDCA was added to the medium, CYP7A1 was reduced to
70% in the single liver biochip. More importantly, during sequential perfusion of intestine and liver, the expression was
further reduced to 40% (Figure 3).
Relative expression of
CYP7A1/ GAPDH
1,2
1
0,8
0,6
*
0,4
0,2
0
Single
perifusion
Sequential
perifusion
Single
perifusion
Control
Sequential
perifusion
CDCA
Figure 3. Expression of CYP7A1 in liver slices in control slices and treated with CDCA with single perifusion and sequential perifusion of intestinal with liver slice. Significant difference toward control is indicated with a * p < 0.05.
These results confirm the intestine-liver signaling pathway and support the hypothesis that FGF15 produced by the
intestine plays a role in the regulation of CYP7A1 in the liver.
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CONCLUSION
This biochip incorporating the sequential perifusion of slices of two different organs is the first model to show in vitro
interorgan signaling using intact tissue. It represents a promising model to contribute to the reduction of animal experiments
and to study these processes in man.
REFERENCES
[1] P. M. van Midwoud, G. M. M. Groothuis, M. T. Merema, E. Verpoorte, Towards improved in vitro systems for ADMETox studies: development of an intestine-liver biochip, Microtechnologies in Medicine and Biology, April 1-3, 2009,
Quebec, Canada.
[2] P. M. van Midwoud, G. M. M. Groothuis, M. T. Merema, E. Verpoorte, Microfluidic biochip for the perifusion of
precision-cut rat liver slices for metabolism and toxicology studies. Biotechnol. Bioeng., 105, pp 184-194 (2010).
[3] T. Inagaki, M. Choi, A. Moschetta, L. Peng, C. L. Cummins, J. G. McDonald, G. Luo, S. A. Jones, B. Goodwin, J. A.
Richardson, R. D. Gerard, J. J. Repa, D. J. Mangelsdorf, S. A. Kliewer, Fibroblast growth factor 15 functions as an
enterohepatic signal to regulate bile acid homeostasis, Cell Metabolism, 2, pp. 217-225 (2005).
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