ARTICLE IN PRESS

Biomaterials 25 (2004) 4683–4690

Fabrication of microstructures in photosensitive biodegradable

polymers for tissue engineering applications
E. Leclerca, K.S. Furukawab, F. Miyatab, Y. Sakaic, T. Ushidab, T. Fujiic,*
a
LIMMS/IIS-CNRS, University of Tokyo, 4-6-1 Komaba Meguro-ku, Tokyo 153-8505, Japan
b
Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-8654, Japan
c
Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba Meguro-ku, Tokyo 153-8505, Japan
Received 23 February 2003; accepted 10 October 2003

Abstract

Combining the MEMS technology and biology requirements for tissue engineering, the fabrication processes of microstructured
chambers and microchannels made in biodegradable photosensitive polymers are presented. The fabrication processes, based on
softlithography are very fast and flexible. Various single and multistepwise microstructures could be achieved using the
biodegradable polymers. Microstructures down to 50 mm, which are suitable for liver reconstructs, could be fabricated. As the
pCLLA acrylate photosensitive polymer has interesting property for implantable bioreactors, that is, its softness, we examined the
ability of various mammalian cells to grow and spread on it. With Hep G2 cells, human umbilical blood vessel endothelial cells
(HUVEC), 3T3-L1 mouse fibroblasts, static cultures could be successfully performed on single stepwise microstructures. Then, by
using this photosensitive biodegradable polymer, a microstructure with simple fluidic channels is fabricated and a perfusion
experiment could be carried out. Both cell cultures and perfusion experiments suggested the possibility to use the present
photosensitive polymer as microfluidic supports for biodegradable bioreactors for implantation applications.
r 2003 Elsevier Ltd. All rights reserved.

Keywords: Microfabrication; Photosensitive biodegradable polymers; Hep G2; HUVEC and 3T3-L1 cell cultures

1. Introduction silicon and PDMS, the cells could be kept living up to 2

Due to the progress in microtechnologies, new
approaches to investigate cells’ interactions and beha-
viours in microenvironment mimicking in vivo situa-
tions are developed as alternative tools for cellular
biology [1,2]. Microtechnologies facilities could be used
to fabricate various types of microbioreactors with
microfluidic network to culture mammalian cells in vitro
[3–6]. Behaviours of Hep G2 cells (Human cancerous
liver cells), mouse fibroblast cells or endothelial cells
were investigated in microfabricated PDMS (polydi-
methylsiloxane) bioreactors [3–5]. This material is a
silicone elastomer, which is biocompatible and exhibits
high gas and oxygen permeability [7–9]. Both properties
were used to support cell culture in the microbioreactors
set in closed perfusion circuits. Silicon bioreactors were
also used to investigate hepatocyte cell culture in
perfusion circuit [6]. With both types of materials,
*Corresponding author. Fax: +81-3-5452-6212.
E-mail address: tfujii@iis.u-tokyo.ac.jp (T. Fujii).
weeks in good conditions as demonstrated by cellular
activity analysis [6]. Those types of microbioreactors
have shown their advantages to perform various toxicity
study [10] or cells’ interaction study [11]. However to
realize in vitro artificial liver tissue for implantation
applications [1,2], those materials are not useful because
they are not degradable.
Recently, to be able to get the tissues for implanta-
tion, several types of biodegradable polymers have been
investigated to fabricate microscaffolds for cell culture
based on a microsyringe deposition or softlithography
techniques [12,13]. Those studies combined successfully
microtechnology and a conventional PLGA biodegrad-
able polymer. The biodegradable microstructures were
used for static endothelial cell culture [14]. Cell growth
and spreading could be observed and demonstrated the
feasibility of cell culture on the microstructured
biodegradable polymers. However, to reach fully large
bioreactors applied to continuous perfusion cell culture,
softlithography techniques applied to this PLGA poly-
mer show several drawbacks. Based on softlithography

0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2003.10.060
 ARTICLE IN PRESS
4684 E. Leclerc et al. / Biomaterials 25 (2004) 4683–4690
fabrication processes, a large bioreactor should be
assembled by superpositioning many layers. This
fabrication technique looks laborious and difficult in
term of layer alignment and probably inconvenient for
the integrity of the bioreactors [15]. To solve this
problem, autonomous 3D fabrication has been applied
as demonstrated in the literature [16]. By one step 3D
fabrication process, a full 3D microfluidic channel
network in PLLA could be fabricated to achieve a
bioreactor. The fabrication process is unique but by a
homemade microsyringe deposition system.
An alternative and new approach of autonomous 3D
fabrication is the photofabrication technique. Since
already developed in microtechnology, the facilities
allowed to fabricate complex and large 3D structures
in photosensitive polymers [17]. However, conventional
PLGA polymer does not contain the required functional
photoreactive material such as acryloil groups. There-
fore, new photosensitive biodegradable polymers have
to be investigated considering compatibility with the cell
culture and with microfabrication techniques.
In the present paper, various fabrication techniques to
fabricate microstructures with photosensitive biode-
gradable polymers are proposed. The softer photosensi-
tive biodegradable polymer will be used for static
cultures of Hep G2, HUVECs (Human Umbilical blood
Vessel Endothelial Cells), and 3T3-L1 cells. The
advantageous feature of this polymer are further
extended by showing the possibility to fabricate a
microfluidic structure for perfusion culture applications.
2. Materials and methods
2.1. Photosensitive polymers
The photosensitive polymers were investigated as
materials of fabrication. The first macromonomer,
which is star-shaped poly-(e-caprolactone (CL)-dl-lac-
tide (LA)) tetraacrylate, was synthesized by a conven-
tional ring-opening reaction of LA using
pentaerythiritol (PE) as an initiator and using Tin 2-
ethylhexanoate as a catalyst and by the addition of
acryloil groups to the end of the four CL-LA chains [18].
This type of photo-crosslinkable macromonomer was
first developed for drug delivery applications, but it has
not been used for tissue engineering. Preliminary
evaluation of the materials formed by UV-irradiation
of the various types of synthesized macromonomers
showed that a macromonomer having a molecular
weight of 5000–10 000 and a composition of CL:LA
=50:50 gives the best characteristics (data not shown).
The macromonomer was designed to be 10 000 in its
molecular weight, and with a blend composition of
PE:CL:LA:Acryloil of 1:50:50:4 (resulting to 25 CL:LA
groups by chains), which allow to get a soft and elastic
material after UV irradiation. The measured molecular
weight was finally: Mw=13 202, the value of Mn was
5727, resulting therefore to ratio Mw/Mn of 2.305. In the
following part, this polymer will be called pCLLA
acrylate.
Another polymer is the commercially available
Kayarad DCPA 60 (Hexaacrylate of caprolactone
modified dipentaerythritol, Cas No. 89800-10-2, Nippon
Kayaku Co. Ltd., Tokyo, Japan). According to the
manufacturer’s information, the molecular weight of
this polymer is 1263, the density is 1.15–1.17, and the
viscosity at 25
C is 900–2000 cps. It was originally tested
for environment application rather than tissue engineer-
ing. As opposed to pCLLA acrylate, this polymer shows
limited elasticity and brittle characteristics after UV
irradiation. In the following part, this polymer will be
called pCLH.
2.2. Initiator and solidification
Upon UV irradiation in the presence of an appro-
priate initiator (1-hydroxycyclohexylphenylketone, Ir-
gacure 184, Chiba Geigy Co., Ltd.), the liquid form
polymers are completely solidified to be yellowish
transparent structures. The mass ratio between the
initiator and the photosensitive polymers was 2:100.
The exposure time will be the function of the energy
supplied by the UV lamp. In our case, the power of the
light was 250 W with wavelength peaks at 345 and
365 nm and the exposure time was 4 min.
2.3. Fabrication methods
2.3.1. Moulding process
The first fabrication method is a moulding process.
The fabrication process of the microstructures is
presented in Fig. 1. For the moulding process, a SU-8
negative master is first made by photolithography
(Fig. 1 i)–iii)) [8,9,19]. Then, a fluorocarbon layer is
deposited by a reactive ion-etching machine (iv). This
layer allowed us to separate more easily the mould
master and the polymer layer. The biodegradable
polymer is deposited on the mould (v), and then is
exposed to the UV light during several minutes (vi).
After the layer is solidified, it could be peeled off from
the mould master (vii).
2.3.2. Direct photolithography process
The second fabrication process is very rapid and
simple as shown in Fig. 2A. It is a direct UV exposure
through a mask pattern to have the desired network. By
spin coating the UV sensitive polymer on the glass
substrate, the thickness of the exposed polymer can be
controlled.
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E. Leclerc et al. / Biomaterials 25 (2004) 4683–4690
i) Glass wafer
v) Polymer deposition
ii) SU-8 coating
vi) UV irradiation
iii) UV exposure
vii) Peel off the solidified polymer
iv) SU-8 development and
fluorocarbon deposition
Fig. 1. Fabrication process of the microstructures in biodegradable
polymer by moulding.
Fig. 2. Fabrication process by UV exposure. (A) using a mask pattern,
and (B) hybrid process with stamping using a PDMS stamp.
2.3.3. Hybrid process
The third fabrication process, see Fig. 2B, combining
the UV exposure with the stamping process [20] was used
to microfabricate single and multistepwise structures in
one layer of photosensitive polymer. After coating the
polymer on a flat glass substrate, a negative stamp of the
desired microstructures made in PDMS is applied on the
polymer. This PDMS stamp was fabricated also by
softlithography [8,9]. Because the PDMS is transparent to
the UV light, it was then possible to solidify the polymer
by UV irradiation through the stamp. The PDMS stamp
could be removed easily from the solidified polymers
resulting in a complete microstructured polymer layer
able to be used for cell culture.
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2.4. Cells and medium preparation
We used Hep G2, human hepatocarcinoma cells, as a
model of liver cells [21] and 3T3-L1 mouse fibroblast
cells, as a model of connective tissue derived cells. The
culture medium for all the routine culture and culture on
microstructures was Dulbecco’s modified Minimum
Essential Medium (DMEM; Nissui Pharm. Co., Ltd.;
Tokyo, Japan) supplemented with 10% fetal bovine
serum (Filtron; Altona, Australia), 25 mm hydroxyethyl-
piperazine-N0 2-ethanesulfonic acid (HEPES; Dojindo,
Kumamoto, Japan), 100 units-penicillin/ml (Wako),
100 mg-streptomycin/ml (Wako) and 0.25 mg-amphoter-
icin B/ml (Sigma).
In some experiments, HUVECs were also used. This
was originally isolated and prepared by Biowhittaker
Inc. (Walkersville, MD, USA) and distributed Sanko
Junyaku Co. Ltd. (Tokyo, Japan). The cells were
routinely cultured in EGM-2 culture medium (Biowhit-
taker Inc.) recommended by the manufacturer. This
culture medium had a low FBS content (5%) and
already contained hydrocortisone, human FGF, human
vascular endothelial cell growth factor (hVEGF),
insulin-like growth factor, human epidermal growth
factor (hEGF), as growth factors. We used cells of less
than 17 cumulative population doubling (CPD).
2.5. Experiments
The experiments of static culture on single stepwise
microstructures made in pCLLA acrylate polymer were
performed. This polymer was chosen because it is the
softer one. A series of four runs of static culture with
Hep G2 cells and three runs with HUVEC and 3T3-L1
cells were performed to demonstrate the ability to
culture cells on this polymer. After fabrication, the
polymer was kept under vacuum conditions for one
night to evaporate remaining toxic solvents, that is,
hexane and diethylether used at the final purification
phase during the synthesis of the polymer. Then,
sterilization by ethanol and 0.03% type I collagen
coating (Nitta gelatin co. Ltd. Osaka, Japan) were
performed. Cells (HepG2, HUVEC or 3T3-L1) were
inoculated with a density of about 3  104 cells/cm2 and
the culture medium was changed periodically every 2 or
3 days.
3. Results
3.1. Microfabricated structures
Fig. 3A shows the view of a mould master made in
SU-8 by classical photolithography process. In Fig. 3B,
the resulting pCLLA acrylate microstructure is shown
after a moulding process done on the mould master
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4686 E. Leclerc et al. / Biomaterials 25 (2004) 4683–4690

Fig. 3. SEM photographs of the fabricated microstructures and mould masters. (A) a SU-8 mould master; (B) microchambers and microchannels on
pCLLA; (C) on PDMS. The resolution is in the same order as (B); (D) a microchannel network on pCLLA; (E) on PDMS; (F) channels fabricated by
direct UV exposure on pCLLA; (G) by moulding process on PDMS; (H) a PDMS stamp; (I) a single stepwise microstructure on pCLH fabricated by
stamping; (J) a multistepwise microstructure on pCLLA fabricated by stamping; (K) PDMS stamp for multistepwise microstructures; and (L) a
multistepwise microstructure on pCLH fabricated by stamping.

shown in Fig. 3A. The replica in biodegradable polymer
fits well the scale of the mould master. Biodegradable
microstructures around 200 mm or above could be
achieved. Indeed the square chamber has a side length
of 400 mm and the channels width is 200 mm, whereas the
depth of the microstructures is about 100 mm. In the field
of the microtechnology, this type of moulding process is
largely applied to achieve PDMS microstructures for
fluidic experiments [8,9] (this process is well known to be
robust and precise). To compare to a PDMS replica, the
same microstructures made in PDMS with the same
mould master are presented in Fig. 3C. The quality of
the resulting channels in both biodegradable polymer
and PDMS is very close at this scale around 200 mm.
Another type of microchannel network in biodegradable
polymer is shown in Fig. 3D whereas the corresponding
PDMS network is shown in Fig. 3E.
By the second fabrication process, which is by direct
UV exposure, the channel shapes obtained were less
precise and less sharp as it is shown in Figs. 3F and G, in
which pCLLA acrylate and PDMS structures are
compared with each other. The drawback of this
method, compared to the first one is the difficulty in
fabricating sharp and high aspect ratio microstructures.
However, 100 mm scales could be achieved as shown in
Fig. 3F.
Then, in Figs. 3H and I, examples of a PDMS stamp
and a microchambers array obtained by the third
fabrication process are shown. The stamp used is the
PDMS layer shown in Fig. 3H. The single step wise
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E. Leclerc et al. / Biomaterials 25 (2004) 4683–4690
microstructure obtained in pCLH polymer in Fig. 3I
obeys perfectly the sizes defined by the stamp. In Figs.
3J and L, finally, biodegradable polymer microstruc-
tures by using the multistepwise stamp shown in Fig. 3K
are also displayed. Fig. 3J is the microstructure made in
pCLLA acrylate whereas Fig. 3L shows the micro-
structure made in pCLH polymer. On the stamp of
Fig. 3H, the smallest characteristic scale is 50 mm. This
characteristic scale appears clearly on the resulting
biodegradable microstructures at the top structure of
microchambers. Thus, the precision of biodegradable
microstructures is limited by the precision of the stamp.
3.2. Morphological observations
Microscopic observations showed good ability of the
polymer for cell cultures, i.e., cell’s attachment in the
initial step, spreading, growth over the microstructures.
As shown in Fig. 4A, tissue-like behaviours of HepG2
cells were observed after 5 days. In Figs. 4B and C, with
the single stepwise microstructures, we could observe
that HepG2 cells and HUVEC cells started to connect
with each other over the stepwise structure to form
larger tissues. These observations were done separately
with both types of cells. In Fig. 4D, 3T3-L1 cells
spreading after 1 week are also shown.
3.3. Cellular analysis
We have compared the Hep G2 cell culture on a
treated culture dish (control) with the one on the present
single stepwise microstructure fabricated in the biode-
gradable polymer. The analysis of the cellular activity
(A) 100 m
(C) 100 m
Fig. 4. Morphological observations of the cells during the culture on a pCLLA acrylate polymer, arrows show the cell’s reorganization. (A) Hep G2
cells after 7 days; (B) Hep G2 cells after 13 days; (C) HUVEC cells after 7 days; and (D) 3T3-L1 cells after 3 days.
4687
was performed by measuring albumin production using
a sandwich-type ELISA [22]. The physiological response
for three runs of the HepG2 cells on the microstructures
is shown in Fig. 5A. Gradual increase of the daily
albumin production illustrated their growth and multi-
plication on the photosensitive polymer. The compar-
ison of those data with the albumin production in the
control experiment contributes to illustrate the possibi-
lity to use this polymer with HepG2 cells.
The daily glucose consumption of the 3T3-L1 cells
was monitored by a glucose analyzer (Glucose analyser 2,
Beckman Instruments Inc., Galway, Ireland) based on the
glucose oxidase method. In Fig. 5B, gradual increase of
the glucose consumption illustrates the cells growth. Both
data demonstrated the ability of utilization of pCLLA
acrylate polymer for both type of mammalian cells.
3.4. Perfusion experiment
In all the present fabrication processes, microstruc-
tures could be realized in the scale range down to 50–
100 mm. The quality of those structures could be
therefore sufficient to be used for perfusion culture.
The liver cell’s characteristic size, such as hepatocytes, is
about 15 mm, whereas microblood-vessel involved in
liver tissues has diameters up to 50–100 mm. The
resolution of fabrication is therefore appropriate for
cell culture. To demonstrate the feasibility of the
pCLLA acrylate polymer, a microfluidic structure with
three different inlets ports and one outlet port was
fabricated. We used the design presented on Fig. 3F.
Each port was a circle hole with a diameter of 1.5 mm.
The width of the main channel is 400 mm and its length is
(B)
100 m
(D) 100 m
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4688 E. Leclerc et al. / Biomaterials 25 (2004) 4683–4690

10 mm. The inlet and outlet channels are 100 mm wide microchannels in the biodegradable polymer are finally
and 3 mm long. The height of the network is 100 mm. covered by a glass substrate as shown in Fig. 6A. A
After exposure and curing of the polymer, inlets and solution of green ink and water was prepared and
outlets were drilled and connected to silicone tubes. injected into the perfusion circuit. During perfusion with
Those tubes are connected to a syringe pumping system the flow rate at 5 ml/min, the fluids flow inside the
for fluid introduction. In this experimental set-up, the channels could be clearly observed under a microscope
flow rate can be controlled from 0.1 to 100 ml/min. The as shown in Fig. 6B. The bonding between the glass and
the polymer layer was sufficiently strong to allow
perfusion at low flow rate.
10
Mean value + SD of the daily albumin

pCLLA, single stepwise,

9
HepG2 (n=3)
production in µg/day/cm2

8
petry control, HepG2, n=3 4. Discussions
7
6 Three fabrication methods were tested with photo-
5 sensitive biodegradable polymers. The resulting scales of
4 the microstructures fabricated by softlithography tech-
3 niques were comparable to the results shown in the
2 literature for non-photosensitive polymers [12–15] in
1 terms of geometry and dimension achieved. In this
0 study, we have fabricated various kinds of microchan-
0 5 10 15 nels at a characteristic scale around 100 mm. This scale
(A) Days of culture satisfies the requirement for the microfluidic environ-
0.25
ment relevant to tissue engineering applications.
Photosensitive biodegradable polymer could be useful
Daily glucose consumption of the 3T3-L1 in

petri control, 3T3-L1 , n=3

3T3-L1, n=3
material due to its rapidness and flexibility in materials
0.2 processing. Indeed, the direct exposure fabrication
process is a fast and simple process to achieve
microstructure composed of multiple feature sizes. The
mg/day/cm 2

0.15
hybrid process is the most appropriate. With this
method, we could fabricate easily, rapidly and repeti-
0.1 tively with the same stamp several microstructured
substrates for cell culture. Thus, fabrication of various
single- and multi-stepwise array of microchambers in
0.05
one polymer layer could be achieved. The moulding
process has shown several drawbacks so far as the
0 demoulding step is concerned. However, by using this
0 1 2 3 4 5 6 7 process with a soft material, as with the pCLLA
(B) Days of culture acrylate, it was possible to get microstructures.
Fig. 5. Activity of the cells during the culture experiments. (A) daily The results of a comparison of the biodegradable
albumin production of Hep G2 cells; and (B) daily glucose consump- microstructures compared to PDMS microstructures
tion of 3T3-L1 cells. (largely used for microfluidic applications) show a

Fig. 6. pCLLA acrylate microfluidic structure used for perfusion experiment. (A) optical microscope view; and (B) perfusion experiment.
 ARTICLE IN PRESS
E. Leclerc et al. / Biomaterials 25 (2004) 4683–4690
similar resolution. With the inlets and the outlet,
perfusion could be successfully operated in the micro-
structure installed in the perfusion circuit. This is very
encouraging for future works with regard to elaboration
with perfusion bioreactor systems and to reach more
complex and functionalized bioreactors. Those results
lead us to expect the possibility to fabricate more
complex 3D structure by using 3D microfabrication
techniques (such as micro stereolithography [17]).
Indeed, as progress has been done, scales in the range
of 200–300 mm can be achieved by the micro stereo-
lithography, which could be useful for tissue engineering
applications. An advantage of this technique is the one
step fabrication process and the possibility to achieve
full 3D structures more easily than other techniques like
softlithography. Compare to conventional porous 3D
PLLA polymer scaffolds, the microfabrication techni-
ques will allow to achieve microfluidic networks for
uniform distribution of culture medium to the cultured
cells, which is reported as a key point for the
construction of bioreactor [23].
One of those photosensitive polymers could be
successfully used with three different cell lines. The
HepG2 cells, HUVEC, and 3T3-L1 cells could attach
and grow on pCLLA acrylate microstructures. The
HepG2 cells could be kept living for 2 weeks with
appropriate sterilization and collagen coating steps. Hep
G2 cells’ attachment, spread, growth and aggregations
were observed by optical microscope. For both HepG2
cells and HUVEC cells, tissues-like arrangement over
the stepwise microstructures could be observed in
several culture experiments.
By the cellular activity analysis, the non-toxicity of
the pCLLA polymer on the cells could be demonstrated.
The daily albumin production has shown no damage in
the function of the cells as long as sterilisation and
removal of toxic solvents are properly performed. This is
an encouraging result in terms of applying this polymer
to larger-scale cultures.
Morphological observations of liver cells such as
hepatocytes or Hep G2 cells have reported the forma-
tion of clusters and 3D spheroid aggregates with
characteristic scales 100–200 mm [24]. In addition, liver
cells in 3D arrangement have shown higher functional
activity in vitro than the monolayer culture. A previous
report dealing with microstructured surfaces composed
of ridges or pillars have shown drawbacks in endothelial
adhesion [25]. In addition, it has been reported that
according to the design of the microstructures, the same
cells could response differently [26,27]. As microholes or
microchambers have been reported in microfluidic
environments to enhance the reorganization of hepato-
cytes [4–6], we have investigated behaviours of the cells
cultured in a microchamber array. With the present
data, we cannot yet conclude that there is the explicit
influence of the stepwise microstructure, which is
4689
supposed to enhance the 3D arrangement of the cells,
onto the cells activity compared to the flat geometry.
However, the difference in albumin production between
flat geometry (culture dish) and the stepwise structure
could be investigated by changing the number of steps in
the microstructures.
5. Conclusions
The fabrication processes of microstructures by using
softlithography with biodegradable photosensitive poly-
mers have been presented. Rapid and flexible fabrication
with the scales down to 50–100 mm can be achieved. The
first fluidic tests shows the possibility to use the
biodegradable polymer with a low flow rate perfusion
system. Furthermore, the pCLLA acrylate has shown
that it could be used to culture mammalian cells. In
future works, those microstructures for perfusion
culture should be examine to realize reorganized tissues
for implantation applications.
Acknowledgements
This work was performed in the framework of
LIMMS, a collaboration initiative between CNRS and
The University of Tokyo, with a support from JSPS.
For the supply of DPCA polymer and advice regarding
the curing conditions, we thank Dr. K. Ishii (Nippon
Kayaku Co. Ltd.). We also thank Mr. M. Tsuru
(Institute of Industrial Science, University of Tokyo)
and Dr. K. Yoshimura (Musashino Chemical Labora-
tory, Ltd.) for supplying the materials.
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