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Biomedical Microdevices 6:3, 219±222, 2004

# 2004 Kluwer Academic Publishers. Manufactured in The Netherlands.

Micro-Patterning of Animal Cells on PDMS Substrates in the


Presence of Serum without Use of Adhesion Inhibitors
Mauris N. De Silva,1 Ravi Desai,2 and
David J. Odde1,2*
1
Department of Chemical Engineering and Materials Science,
University of Minnesota
E-mail: desilva@mozart.cems.umn.edu
2
Department of Biomedical Engineering, University of Minnesota
E-mail: oddex002@umn.edu

Key Words. micropatterning, cell patterning, PDMS, microcontact has become widely used for micropatterning substrates
printing
with biomolecules permissive to cell attachment and
growth (Chang et al., 2003; Chen et al., 1997; Lopez et
al., 1993; Wilbur et al., 1994). In this technique,
molecules of interest are ®rst adsorbed onto a PDMS
Introduction stamp and then transferred to a chemically activated
substrate by direct contact of the substrate with the
Micro-scale cell patterns are commonly obtained by
stamp. Regions that are not stamped are then treated
culturing cells on a substrate having a cell adhesive
with another molecule that inhibits cell adhesion, such
molecule pattern. Compliance to the pattern is ensured
as albumin (Wheeler et al., 1999), polyethylene glycol
by coating adjoining regions with a cell non-adhesive
(Irimia and Karlsson, 2003; Wheeler et al., 1999), or
molecule (e.g., polyethylene glycol) that inhibits
pluronic (Gopalan et al., 2003). We used the micro-
protein absorption or cell adhesion (Kleinfeld et al.,
contact printing technique to pattern substrates with
1988; Wheeler et al., 1999). Current techniques for
adhesive biomolecules, as is the usual practice, but then
preparing patterned substrates require complex surface
directly plated the cells without applying any inhibitor
chemistry to immobilize both cell adhesive molecules
of cell adhesion to the unstamped regions prior to cell
and inhibitory molecule patterns on the cell culture
plating. To achieve this simpli®cation, we used PDMS
substrate surface. Furthermore, the presence of serum
as the substrate that was spin-coated onto petri dishes.
in the culture medium, although bene®cial to cell
Once stamped with an extracellular matrix component
growth, tends to foul the non-adhesive surface
(either laminin or collagen), these dishes were used for
resulting in pattern degradation over time (Branch et
cell patterning without further modi®cation. The PDMS
al., 2001). Here, we describe a method that allows
area on the substrate that didn't contact the stamp was
micro-scale surface patterning of living animal cells
free of the adhesion-promoting biomolecule, and acted
without using complex surface chemistry or adhesion
as an inhibitory region for cell attachment, while the
inhibitors. The method uses polydimethylsiloxane
stamped region acted as a region for promoting cell
(PDMS) stamps to create laminin (or collagen) patterns
adhesion due to the adsorption of the adhesive
on PDMS substrates to permit cell attachment and
molecules on PDMS substrate during the microcontact
growth, and exploits the natural tendency of PDMS to
printing process (Figure 1). These micropatterned
inhibit cell adhesion. The method was applied to
PDMS substrates were used to culture embryonic
embryonic chick forebrain cells, embryonic chick
chick forebrain cells, embryonic chick spinal cord
spinal cord cells, LLCPK1 epithelial cells, and
cells, LLCPK1 epithelial cells, and HUVEC in serum
human umbilical vein endothelial cells (HUVEC).
containing media to obtain micropatterns of cells.
Within 1±2 days, cells were con®ned to the areas
Laminin was used to enhance the adhesion of embryonic
that contained cell adhesive molecules and avoided the
chick forebrain cells, embryonic chick spinal cord cells,
unprinted PDMS regions. Cells remained patterned for
and LLCPK1, while collagen was used to enhance the
up to a week in the presence of serum. The method
adhesion of HUVEC. Patterns were formed within 1±2
provides an inexpensive and simple method for cell
patterning in cell biological studies and biotechnolo-
gical applications.
Among various techniques available for micro-scale
patterning of cells, the microcontact printing technique *Corresponding author.

219
220 De Silva, Desai and Odde

Fig. 1. Schematic for method of direct patterning of animal cells by PDMS-PDMS stamping. A PDMS stamp coated with an adhesion-promoting
biomolecule is used to directly pattern a PDMS-coated Petri dish. Cells are then cultured on the dish. The stamped regions of the dish are
permissive for cell adhesion, while the unstamped regions are non-permissive for cell adhesion even in the presence of serum in the medium for
several days.

days and cells remained viable for approximately a From Figure 2, it is clear that the concentration of
week (Figure 2). adhesion promoter on the stamped portion is adequate to
Because our technique does not use inhibitory permit cell adhesion for a wide range of cell types. While
molecules, it is much simpler to implement than previous in serum-containing media, adhesion promoting bio-
microstamping methods, all of which require the molecules in the serum (such as ®bronectin) could
immobilization of an inhibitory molecule to suppress potentially adsorb onto the unstamped surface and permit
cell adhesion away from the patterned biomolecule. The cell adhesion, thus destroying the initial pattern.
most common inhibitory molecules used to inhibit cell However, cell adhesion on the unstamped region was
adhesion are typically either expensive or not commer- very limited and so we conclude that the surface density
cially available, and their immobilization requires of the adhesion molecules adsorbed from the media is
multiple step procedures. Commonly used immobiliza- much lower than that transferred via the microstamp.
tion methods for PEG are silanization (Irimia and Thus, the natural tendency of PDMS to resist protein
Karlsson, 2003) and chemical modi®cation of the adhesion is overcome by microstamping, but not by
substrate followed by PEGylation (Branch et al., 2000; adsorption from an aqueous solution.
Chen et al., 1997; Wheeler et al., 1999). Since these The method presented here for micro-scale surface
methods of assembling non-permissive biomolecules patterning of biological molecules achieves long-lived
employ complex surface chemistry, some commonly cell patterns in the presence of serum without
observed problems are that they take an extensive employing complex surface chemistry or using adhesion
amount of time to implement in a lab and are not easy inhibitors. As micropatterning expands into commercial
to reproduce. By comparison our technique does not biosensor applications and an increasing range of basic
require the use of inhibitory molecules or any surface biological research applications, the ability to achieve
modi®cation methods and employs only a single patterns simply and cheaply will become increasingly
microcontact printing step. Therefore, it becomes less important. The present method advances the art in both
complicated and easier to reproduce. Also, our method is respects, and so provides an improved tool for cellular
less expensive as the need for functionalized PEG bioengineers.
molecules or other reagents is obviated.
An additional advantage of the method is that, unlike
existing methods, it permits maintenance of the cell Methods
patterns in the presence of serum, which will expand the
range of possible applications. In our method, laminin Fabrication of PDMS substrates
and collagen act as adhesion promoters and the A 10 : 1 …w : w† ratio of elastomer monomer : curing
unstamped PDMS surface acts as an adhesion inhibitor. agent (Sylguard 184 Silicone Elastomer, Dow Corning)
Micro-Patterning of Animal Cells on PDMS Substrates 221

Fig. 2. Micro-scale cell patterns. The cell patterns were obtained on PDMS substrates patterned with laminin for (a) LLCPK1, (b) chick forebrain
cells, and (c) chick spinal cord, and collagen solution for (d) HUVEC. Scale bar ˆ 100 mm.

was thoroughly mixed and allowed to cure for 30 minutes SDS for 5 minutes, dipped in DI H2O, and blown dry
at room temperature. Two hundred and ®fty milligrams with a stream of N2 for 45 seconds (Chang et al., 2003).
of the elastomer were poured into a 35 mm petri dish and Fifty microliters of 20 mg/ml laminin (Natural mouse,
the dish was then spun at 3,000 rpm for 30 seconds with a Invitrogen Corporation) solution or 20 mg/ml laminin
spinner to create an elastomeric ®lm a few micrometers solution ‡ 5% (vol/vol) FITC-laminin solution ( pre-
thick on the surface of each dish. PDMS substrates were pared using FITC-conjugation kit (Sigma) and stock
allowed to cure for at least ®ve days at room temperature laminin) or 0.2% aqueous gelatin solution was pipetted
following spin coating to ensure full mechanical directly onto the stamp face. Stamps were incubated at
strength. room temperature and humidity with the laminin solution
for 1 hour, and the gelatin solution for approximately
PDMS-PDMS stamping 5 minutes. Excess adhesion-promoting solution was
Molds for PDMS stamps were made by patterning pipetted off and stamps were blown dry with a stream
photoresist 1813 (Shipley) on Si wafers. PDMS solution of N2 for 45 seconds. Loaded stamps were immediately
was made as described above, and poured into the mold, inverted onto a PDMS substrate for 1 hour at room
and cured at room temperature for approximately three temperature and humidity; in some cases a 25 g weight
days. PDMS stamps were removed from their mold, was placed on top of the stamp for the duration of the
sonicated in 10% SDS for 5 minutes, immersed in 10% stamping period.
222 De Silva, Desai and Odde

Cell culture Invitrogen Corp) with 5% FBS and atibiotic/antimycotic


solution ( pen, strep and amphotericin B).
HUVEC culture. HUVEC were purchased from Vec
Technologies, New York, and were cultured according to
the manufacturer's standards in Medium 199 (Gibco)
containing 15% fetal bovine serum (Gibco), 40±50 mg/ Acknowledgments
ml EndoGro (Vec Technologies), 10 U/ml heparin
(Sigma-Aldrich), 30 mg/ml sodium pyruvate (Gibco), The authors would like to thank Yaakov Nahmias,
0.7 mg/ml L-glutamine (Gibco), 100 U/ml penicillin/ Abhinav Arneja, and Andrew Bicek for their advice in
streptomycin (Gibco). HUVEC and LLCPK1 cell culture and the staff of the
Nano Fabrication Center of the University of Minnesota,
Dr. Babak Ziaie, and Tingrui Pan for their advice in
Chick forebrain cell culture. Chick forebrain cell fabrication technologies. Funding for this project was
culture was made similar to the method described in partially provided by NSF-BITS Grant No. EIA0130875
Baldi et al. (2003). Brie¯y, cerebral hemispheres of E7± and through the Microfabricated Neural Networks
E8 chick embryos were dissected and meningeal Interest Group by the Biomedical Engineering Institute
membranes were removed. The cerebral tissue was at University of Minnesota.
then incubated at 37  C in 0.25% trypsin solution (Gibco)
for 15 minutes, after which cells were resuspended in
media (1 : 1 DMEM:F12 supplemented with 10% fetal
bovine serum, 100 U/ml penicillin, 100 mg/ml strepto- References
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