a simple hydrophilic treatment of su-8 surfaces for

A SIMPLE HYDROPHILIC TREATMENT OF SU-8
SURFACES FOR CELL CULTURING AND CELL
PATTERNING
aw2328.pdf
Z. Wang, M. Stangegaard, M. Dufva, J.P. Kutter, and A. Wolff
Department of Micro and Nanotechnology, Technical University of Denmark, Denmark
Abstract: SU-8, an epoxy-based photoresist, widely used in constitution different PTAS
systems, is incompatible with mammalian cell adhesion and culture in its native form. Here,
we demonstrate a simple, cheap and robust two-step method to render a SU-8 surface
hydrophilic and compatible with cell culture. The contact angle of SU-8 surface was
significantly reduced from 90q to 25q after the surface modification. The treated SU-8
surfaces provided a cell culture environment that was comparable with cell culture flask
surface in terms of generation time and morphology.
Keywords: SU-8, Surface modification, Contact angle, Cell culture
SU-8, an epoxy-based negative photoresist, is widely used for fabricating microstructures in
various PTAS devices because of its excellent chemical stability and optical properties.
However, since the SU-8 surface is hydrophobic, cell adhesion and culturing is hampered in
SU-8 based microsystems.
Human epithelial cells, such as HeLa cells [1], preferably adhere and grow on hydrophilic
surfaces [2]. Hydrophilic surfaces can be generated e.g. by coating SU-8 surfaces with
fibronectin [3] or hydrophilic polymer materials [2] to allow culturing of cells. However,
such dynamic coatings may not be stable over time. Chemical treatments, on the other
hand, have the potential to modify the SU-8 surface properties for extended periods [4].
Here, we report a simple, cheap and robust chemical treatment to render a SU-8 surface
hydrophilic.
Normally, the epoxy moieties on the SU-8 surface do not react with standard corrodents
(e.g., THF, HCl or HNO3), resulting in the pronounced chemical stability of SU-8 [5].
However, with the catalyst ceric ammonium nitrate (CAN, (NH4)2Ce(NO3)6), the epoxy
rings can be opened and reacted with HNO3 [6] (Fig 1).
Fig 1. Reaction scheme of the two-step SU-8 surface modification: 1: The epoxy ring on
the SU-8 surface is believed to be opened by treatment with 1 M HNO3, catalyzed by 0.1 M
CAN at 50qC for 1 hour; 2: The nitrate radical on the modified SU-8 surface was then
reacted with the amino group of ethanolamine (0.1 M) in 0.1 M sodium phosphate buffer
(pH = 9.0) for 20 minutes at 50qC.
9th International Conference on Miniaturized Systems for Chemistry and Life Sciences
October 9-13, 2005, Boston, Massachusetts, USA
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Cell density (cells/mm )
After this first CAN-HNO3 treatment, the nitrate radical on the opened epoxy ring can react
further with the amino group of ethanolamine. The contact angle of water on the differently
treated SU-8 surfaces was used to represent the changes in hydrophobicity (Fig 2A). The
contact angle on the SU-8 surface was reduced from 95° to 45° by the CAN-HNO3
treatment. This indicates that hydroxyl groups were formed on the SU-8 surface. After the
second (ethanolamine) treatment, the contact angle on the SU-8 surface was further reduced
to 25°. However, the second treatment is an ionic reaction, and hence reversible: after 2
months of storage, a contact angle of 50° was observed.
Contact angle
100
A
80
60
40
20
0
a
b
c
d
Differently treated SU-8 surfaces
a
b
c
e
1000
800
600
B
400
200
0
1
2
3
4
Day
Fig 2. Contact angle (A) and biocompatibility (B) of differently treated SU-8 surfaces. The
letters on the x-axis refer to the following SU-8 surfaces: (a). Untreated; (b). Treated with
CAN-HNO3; (c). Treated with CAN-HNO3 and ethanolamine; (d). Treated with CANHNO3 and ethanolamine and then stored for 2 months; (e). Control culture flask surface A:
The hydrophobicity of differently treated SU-8 surfaces was determined by measuring the
contact angle of water. B: For the biocompatibility test, differently treated SU-8 surfaces
were immersed in standard cell culture media (RPMI 1640 media supplemented with 10%
Fetal Bovine Serum (FBS), 1000 U/mL penicillin and 1 mg/mL streptomycin) in cell
culture flasks and seeded with HeLa cells. The samples were incubated for 4 days at 37°C
in an atmosphere containing 5% CO2.
For testing the biocompatibility, HeLa cells were grown on differently treated SU-8
surfaces for 4 days. The differently modified SU-8 surfaces resulted in different attached
cell densities and cell growth rate (Fig 2B). Untreated SU-8 had a very low cell density
throughout the experiments, as the cells only attached the surface, and did not proliferate
(generation time: 147±56 hours). CAN-HNO3 treated SU-8 had a significantly higher cell
density and lower generation time (36±0.7 hours). After the second treatment
(ethanolamine), the cell density were further increased and generation time reduced (32±0.1
hours), and comparable with that on the control culture flask (32±0.9 hours). The results
indicate that the cell generation time decreases with the hydrophobicity of the culturing
surface. Finally, HeLa cells were cultured on untreated SU-8 surfaces with hydrophilic
patterns (Fig 3). The images show that HeLa cells changed their morphologies to fit onto
the hydrophilic SU-8 patterns, suggesting that HeLa cells actively avoided the hydrophobic
surface of untreated SU-8.
9th International Conference on Miniaturized Systems for Chemistry and Life Sciences
October 9-13, 2005, Boston, Massachusetts, USA
746
Fig 3. HeLa cells cultured over night on SU-8 surfaces with hydrophilic patterns. The
hydrophilic patterns were created by standard photolithography of AZ5214e photoresist on
top of a 5 µm SU-8 layer. Then, the whole glass wafer was dipped into HNO3 with CAN for
the treatment. Finally, the photoresist was lifted off in acetone (4 minutes ultrasonic
agitation.) A: HeLa cells growing on an array of 50 Pm u 50 Pm squares; B: HeLa cells
growing on concentric circles. A change in the cell morphology was observed during
culturing. It appeared as though the cells would change their morphologies to fit on to the
hydrophilic SU-8 patterns rather than display their normal morphologies and hence be in
contact with the untreated SU-8.
In conclusion, a two-step chemical treatment for modification of SU-8 surfaces was
developed. The treated SU-8 surface shows good biocompatibility for cell culturing, and
indicates a possible route to integrate cell culture functionalities in SU-8 based PTAS
devices. The treatment can be used to create hydrophilic patterns on the SU-8 surface for
defining localized cell growth.
References:
[1] G. O. Gey, W. D. Coffman, and M. T. Kubicek, Cancer Research, 12, 264-265, 1952.
[2] S. Bouaidat, C. Berendsen, P. Thomsen, S.G. Pederson, A. Wolff, and J. Jonsmann,
PTAS2004, 2, 106-108, 2004.
[3] N. D. Gallant, J. R. Capadona, A. B. Frazier, D. M. Collard, and A. J. Garcia,
Langmuir, 18, 5579-5584, 2002.
[4] M. Nordstrom, R. Marie, M. Calleja, and A. Boisen, J. Micromech. Microeng. 14,
1614-1617, 2004.
[5] Y. Song, C.S.S.R. Kumar, and J Hormes, J. Micromech. Microeng. 14, 932-940, 2004.
[6] N. Iranpoor, P. Salehi, Tetrahedron, 51, 909-912, 1995.
9th International Conference on Miniaturized Systems for Chemistry and Life Sciences
October 9-13, 2005, Boston, Massachusetts, USA
747

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