THREE-DIMENSIONAL (3D) HOLLOW
POLYMERIC MICROSTRUCTURES FOR SHEARPROTECTING CELL CONTAINERS WITHIN
MICROFLUIDIC CHANNEL
1
Sung-Hoon Lee,1 Hye-Sung Cho,1 Chan-Ick Park,1 and
Kahp-Yang Suh1
School of Mechanical and Aerospace Engineering and Institute of Advanced
Machinery and Design, Seoul National University, Seoul 151-742, Korea.
ABSTRACT
We present a simple soft lithographic method to integrate highly optimized,
polymeric cell containers within a microfluidic channel for cell docking and shear
protection. The 3D hollow cell containers were generated by partial molding of polymethyl methacrylate (PMMA) through a solvent-assisted capillary molding technique. The molded polymeric microstructures were used to capture budding yeast
cells, Saccharomyces cerevisiae, within microfluidic channel and the response of
yeast cell was observed upon stimulation by the mating pheromone (α-factor) or
high osmolarity (KCl) by monitoring the expression of green fluorescent protein
(GFP) over time.
KEYWORDS: Hollow microstructure, Cell container, Shear-protection
INTRODUCTION
The ability to capture living cells inside a microfluidic channel in a scalable,
shear-protecting manner is important for the development of high-throughput cell
screening and biological analysis. In
microfluidic channel environments,
the medium flows over the adhered or
suspending cells while markedly influencing cell growth, cellular function, and viability regardless of the
cell type [1]. It is therefore desirable to
take shear damage into account when
designing and fabricating microchannel or bioreactor. This study addresses
a method of forming 3D bottle-shaped,
hollow polymeric microstructures inside a microfluidic channel for potential shear-protecting cell containers.
Figure 1. Schematic of fabricating 3D
EXPERIMENTAL
The fabrication of 3D hollow hollow polymer structures inside a micromicrostructure was performed by fluidic channel for shear-protecting cell
partial molding technique based on solvent-assisted capillary force lithography [2].
Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences
October 12 - 16, 2008, San Diego, California, USA
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This method typically consists of three steps: placing a patterned PDMS mold on the
surface of a drop-dispensed polymer film (~20 ㎕), applying slight pressure (~ 10 g/
㎠), and then allowing the mold to absorb solvent and the substrate remain undisturbed for a period of time until the structure solidifies after evaporation of the solvent. After forming hollow polymeric microstructures, the yeast cells were captured
inside the structures under stationary conditions (sedimentation) and analyzed under
a fluorescent microscope. The SEM images (bottom) show an example of hollow
PMMA microstructure (Fig. 1a) and its cross-sectional view (Fig. 1b).
RESULTS AND DISCUSSION
A completely molded microstructure can be easily fabricated using a relatively high
polymer concentration (> 10 wt% in toluene) or a large film thickness (~ > 8 ㎛),
which is needed to facilitate mass transport from the substrate (Fig. 2a). In contrast, a
bottle-shaped, hollow structure was
formed for a relatively low concentration (< 10 wt% in toluene) or a small
film thickness (~ < 750 nm). In this
case, the polymer film under the void
space of PDMS mold was nearly consumed as judged by the sharp boundary
at the polymer/substrate interface (Fig.
2b).
For fabricating 3D hollow microstructure, two conditions need to be
satisfied: a PDMS mold should have a
bottle-shaped
geometry
(slanted
walls) with an acute angle at the corner. This geometry gives rise to the
pinning of meniscus at the corner,
thereby leading to progression of the
liquid
front without merging. In addiFigure 2. (a, b) Fabrication of completely
tion,
the
polymer solution should have
(a) and partially (b) molded cylindrical
good
affinity
to both glass substrate
PMMA microposts. (c-f) Control of strucand
PDMS
wall.
This condition enture dimen-sion of pyramidal microposts
sures
fast
capillary
rise of the solution
using different amount of polymer solualong
the
wall
while
preventing the
tion: ~ 5 ㎕ in (c), ~ 10 ㎕ in (d), ~ 15 ㎕
formation of a uniformly curved mein (e), and ~ ~ 20 ㎕ in (f).
niscus.
To evaluate the shear-protecting ability, we measured changes in fluorescent intensities of the mating and high osmolarity signaling pathways for docked and nondocked cells, respectively. In triggering salt-responsive SH113 cells with 1M KCl,
the fluorescence intensity of docked cells increased continuously, while that of nondocked cell decreased after 120 min (Fig. 3a). In the case of mating SH129 cells
with 10 µM α-factor, the fluorescence intensity of docked cell was almost main-
Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences
October 12 - 16, 2008, San Diego, California, USA
1130
tained at the initial level, while the fluorescence intensity of non-docked cell
dropped drastically with a relatively short period of exposure time (Fig. 4b).
Figure 3. (a) The cell responses
triggered with high osmolarity. (b)
The cell responses triggered with
mating pheromone. (c) The
change of normalized fluorescence
intensities as a function of exposure time to medium flow when
triggered with high osmolarity. (d)
The change of normalized fluorescence intensities as a function of
exposure time to medium flow with
various dimensions (height/entra
nce diameter)when triggered with
mating pheromone. (e) Evaluation
of shear-protecting ability of 3D
hollow cell container. (f) Comparison of the shear protecting
ability of 3D hollow cell container
with that of vertical microwell.
CONCLUSIONS
In summary, we have presented a simple method for fabricating shear-protecting
cell containers integrated within a microfluidic channel. It is envisioned that the microfabrication method presented here could serve as a simple, inexpensive way to
fabricate microfluidic device for high throughput analysis of cells under different
shear flows or other signaling cues with cell reservoirs of different geometry.
ACKNOWLEDGEMENTS
This work was supported by the Grant-in-Aid for Next-Generation New Technology Development Programs from the Korea Ministry of Knowledge Economy
(No.10030046). This work was also supported by the Intelligent Microsystem Center (IMC; http://www.microsystem.re.kr), which carries out one of the 21st century's
Frontier R&D Projects sponsored by the Korea Ministry of Knowledge Economy.
REFERENCES
[1] Glenn M. Walker, Henry C. Zeringue and David J. Beebe, “Microenvironment
design considerations for cellular scale studies.” Lab Chip, 4, pp. 91-97 (2004).
[2] Y. S. Kim, K. Y. Suh, and Hong H. Lee, “Fabrication of three-dimensional microstructures by soft molding.” Appl. Phys. Lett. 79, pp. 2285-2287 (2001).
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October 12 - 16, 2008, San Diego, California, USA
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