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Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused 3DPorous Polymer Scaffold for Liver Tissue Engineering
Hemmingsen, Mette; Muhammad, Haseena Bashir; Mohanty, Soumyaranjan; Wolff, Anders; Emnéus,Jenny; Aspegren, Anders; Dufva, Martin
Publication date:2014
Link back to DTU Orbit
Citation (APA):Hemmingsen, M., Muhammad, H. B., Mohanty, S., Wolff, A., Emnéus, J., Aspegren, A., & Dufva, M. (2014).Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused 3D Porous Polymer Scaffold forLiver Tissue Engineering. Poster session presented at 3rd International Conference on Tissue Science &Regenerative Medicine, Valencia, Spain.
Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a
Perfused 3D Porous Polymer Scaffold for Liver Tissue Engineering
*Technical University of Denmark, 2800 Kgs. Lyngby, Denmark, **Cellectis AB, Gothenburg, Sweden.
Website: www.nanobio4trans.eu e-mail: [email protected]
Mette Hemmingsen*, Haseena Bashir Muhammad*, Soumyaranjan Mohanty*, Anders Wolff*,
Jenny Emneus*, Anders Aspegren**, Martin Dufva*
Introduction and Aim
A huge shortage of liver organs for transplantation has motivated the research field of tissue engineering to develop bioartificial liver tissue and even
a whole liver. The goal of NanoBio4Trans is to create a vascularized bioartificial liver tissue, initially as a liver-support system. Due to limitations of
primary hepatocytes regarding availability and maintenance of functionality, stem cells and especially human induced pluripotent stem cells (hIPS
cells) are an attractive cell source for liver tissue engineering. The aim of this part of NanoBio4Trans is to optimize culture and hepatic differentiation
of hIPS-derived definitive endoderm (DE) cells in a 3D porous polymer scaffold built-in a perfusable bioreactor. The use of a microfluidic bioreactor
array enables the culture of 16 independent tissues in one experimental run and thereby an optimization study to be performed.
Funding provided by EU Program HEALTH.2012.1.4-2 [Medical technology for transplantation and bioartificial organs], Contract:304842
Bioreactor Array System for Tissue Culture
Our approach for engineering liver tissue is to
culture and differentiate hIPS-derived DE cells
in a 3D porous polymer scaffold housed in a
perfusable bioreactor to ensure supply of
oxygen and nutrients and removal of waste.
Single bioreactor
Porous polymer scaffold
Bioreactor array
Outlet vials
LEGO® motor
8 channel
micropumps
Inlet vials
Pressure inlet to avoid
bubble formation
Issues at Flow Conditions
Time
Conc. of
added
factors
At Flow Conditions
Time
Conc. of
added
factors
At Static Conditions
.
Concentration of Added
Signaling Factors
One important parameter when optimizing conditions for flow cultures is the flow rate. The flow rate is
a balance between ensuring sufficient supply of nutrients and oxygen, but at the same time avoiding
to much shear stress onto the cells and removal of cell-to-cell signaling factors. Besides testing
different flow rates, we apply different scaffold designs with a different flow profile. Another issue to
consider is the concentration of added signaling factors. At flow the concentration of added factors is
constant, while at static conditions in a batch culture, the concentration decreases between each
exchange of medium. Thus, a lower concentration than the one optimized for batch cultures might be
better at flow conditions.
Gene Expression of Liver Markers
0
0.5
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1.5
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Ge
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[r.
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ALB
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Gen
e ex
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AFP
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e ex
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.]
CYP3A5
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0.0005
0.001
0.0015
0.002
0.0025
0.003
0.0035
0.004
Gen
e ex
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r.u
.]
CYP3A7
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exp
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[r.
u.]
HNF4A
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0.0005
0.001
0.0015
0.002
0.0025
0.003
0.0035
0.004
0.0045
Gen
e ex
pre
ssio
n [
r.u
.]
CAR
0
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ne
exp
ress
ion
[r.
u.] ALB
0
500
1000
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Ge
ne
exp
ress
ion
[r.
u.] AFP
0
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0.1
0.15
0.2
0.25
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0.35
0.4
Ge
ne
exp
ress
ion
[r.
u.] CYP3A5
0
0.00005
0.0001
0.00015
0.0002
0.00025
Ge
ne
exp
ress
ion
[r.
u.] CYP3A7
0
5
10
15
20
Ge
ne
exp
ress
ion
[r.
u.] HNF4A
0
0.005
0.01
0.015
0.02
0.025
Ge
ne
exp
ress
ion
[r.
u.] CAR
A) Conditioned medium collected from a parallel static
batch culture increased the gene expression of liver
markers. Analysed at day 9 and day 14 after the DE stage.
Similar gene expression level of the hepatocyte nuclear transcription factor 4α was observed at
perfusion cultures compared to conventional static cultures, whereas a decreased expression was
seen for the transcription factor CAR and the CYP enzymes CYP3A5 and CYP3A7. Furthermore,
expression of albumin and α-fetoprotein was almost knocked down. However, expression of most of
the markers was increased by the use of A) conditioned medium or B) medium with a two times lower
concentration of the added signalling factors than those optimized for conventional batch cultures.
No
rma
l m
ed
ium
C
on
ditio
ne
d m
ed
ium
B) Differentiation medium with a two times lower concentration of
added signalling factors than those optimized for conventional
batch cultures (2x dil. medium) resulted in increased expression of
most liver markers at day 14 after the DE stage.
Conclusion
The results suggest that the flow conditions affect cell-to-cell signalling necessary for liver differentiation and/or functionality, as well as the
concentration of added signalling factors in the differentiation medium has to be adapted to the different environment at flow culture with
constant renewal of the culture medium.
Cell morphology
A hepatocyte-like cell morphology was obtained with
polynucleated cells and ”bile-canaliculi”-like structures
(shown by the red arrows). Imaging at day 14 of
differentiation after DE stage. Live stained cells A) and
Höchst stained cells B) in the 3D scaffold. C) Phase contrast
image of differentiated cells in a 2D perfusion chamber.
A)
B)
C)
Inlet
Outlet
Flow Rate
1. Supply of nutrients 2. Removal of waste
1. Shear stress 2. Removal of cell-
cell signaling factors
6 mm
5 mm
The bioreactor array enables culture/differentiation
of 16 independent tissues in one experimental run.