Photoactivation of a substrate for cell adhesion under standard fluorescence microscopes

Article abstract of DOI:10.1021/ja044684c

Cell-culturing substrates where cell adhesion can be switched on by external stimuli during cell cultivation are useful scaffolds for tissue engineering, cell-based drug screening, and fundamental cellular studies. Here, we show a new strategy for photoac

Full text of DOI:10.1021/ja044684c

Published on Web 11/26/2004
Photoactivation of a Substrate for Cell Adhesion under Standard
Fluorescence Microscopes
Jun Nakanishi,† Yukiko Kikuchi,† Tohru Takarada,*,† Hidekazu Nakayama,‡ Kazuo Yamaguchi,‡ and
Mizuo Maeda*,†
Bioengineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, and Department of Materials
Science, Faculty of Science, Kanagawa UniVersity, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan
Received September 2, 2004; E-mail: ttkrd@riken.jp; mizuo@riken.jp
Dynamic control of cell adhesion onto substrates during cell
cultivation is important for tissue engineering,1,2 cell-based drug
screening, and fundamental cellular studies.3-5 To realize such dy-
namic control, several functional substrates have been developed,
where cell adhesion can be controlled in response to external
stimuli, such as heat,6 voltage,7-10 light,11-13 and enzymatic activ-
ities.14 However, their dynamically controllable regions are prede-
termined unless additional equipment is used to apply external
stimuli.
Here, we show a new method for photoactivation of any regions
of a substrate for cell adhesion under standard fluorescence
microscopes. The strategy is based on a silane coupling agent having
a photocleavable 2-nitrobenzyl group, (1-(2-nitrophenyl)ethyl-5-
trichlorosilylpentanoate, NPE-TCSP),15 and on two commonly used
proteins that either prevent or promote cell adhesion. The procedure
was as follows (Figure 1a). A glass coverslip chemically modified
with NPE-TCSP was coated with bovine serum albumin (BSA) in
order to make the surface inert to cell adhesion. The coverslip was
then irradiated, causing the 2-nitrobenzyl group to be photocleaved
and BSA to be dissociated from the surface. The addition of
fibronectin, a protein that promotes cell adhesion by the integrin-
mediated interaction, led to its selective adsorption at the vacancy
Figure 1. Schematic representations of photoactivation of cell adhesion.
(a) UV-directed replacement of BSA with fibronectin changed the surface
of BSA, causing the region to become cell-adhesive. The 2-ni-
trobenzyl group is a common photocleavable protecting group for
surface modification16-18 or instantaneous release of physiologically
active compounds,19 which is cleaved by UV light (∼365 nm) from
a mercury lamp equipped on standard fluorescence microscopes
(Figure 1b). This feature allowed remote control of cell adhesion
by simple operation of the widely available equipment.
from a state that prevents cell adhesion to a state that promotes cell adhesion.
(b) Experimental setup for irradiation under a standard fluorescence
microscope.
We first evaluated whether cells selectively attached to the
irradiated region on the substrate. A glass coverslip chemically
modified with NPE-TCSP was coated with BSA and irradiated over
a circular region (Figure 2a) for 2.5 min under a fluorescence
microscope. This irradiation changed the contact angle of the
substrate from 65 to 46°, and the change in wettability was nearly
completed in this irradiation time (Figure S1 in the Supporting
Information). After incubation of the substrate with fibronectin for
30 min, HEK293 cells selectively attached to the circular region
in 2 h (Figure 2b). The attached cells were confined to grow within
the region and became confluent at 63 h (Figure 2c). Almost the
same results were obtained with other cell types, such as COS7
and NIH3T3 (Figure 2d,e). On the other hand, no cell-adhesive
region was formed on a glass coverslip modified with a nonpho-
tolabile 2-(carbomethoxy)ethyltrichlorosilane (Figure S2 in the
Supporting Information). Moreover, the addition of fibronectin is
essential for the formation of cell-adhesive spots because the cell
adhesiveness to the irradiated area is very weak when fibronectin
Figure 2. Formation of cell-adhesive spots in response to light. (a) UV-
irradiated region. (b and c) Phase-contrast images of HEK293 cells 2 h (b)
and 63 h (c) after seeding. (d and e) Phase-contrast images of COS7 cells
(d) and NIH3T3 cells (e) 18 h after seeding.
is omitted from the experimental procedure (Figure S3 in the
Supporting Information).
Exchange of the surface protein from BSA to fibronectin was
confirmed by immunofluorescence microscopy. BSA dissociated
from the surface by UV irradiation, and the following addition of
fibronectin resulted in its selective adsorption onto the irradiated
region (Figure S4 in the Supporting Information). The possible
mechanism for the dissociation of BSA is as follows. BSA is
adsorbed on the surface in contact with the 2-nitrobenzyl groups.
The photocleavage of the 2-nitrobenzyl groups allows BSA to
diffuse into the bulk, accompanying the cleaved protecting groups.
Moreover, the photocleavage reaction changes the chemical species
from the hydrophobic nitrobenzyl ester to the polar carboxyl group
at the very surface. This increase in the hydrophilicity may diminish
† RIKEN.
‡ Kanagawa University.
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10.1021/ja044684c CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. Cell attachment to array-patterned spots corresponding to
photomasks inserted at the location of the field diaphragm of a fluorescence
microscope. (a and c) Fluorescence images of glass coverslips painted with
a fluorescent pen, which were irradiated using corresponding photomasks.
(b and d) Phase-contrast images of HEK293 and COS7 attached on the
substrates 18 and 2.5 h after seeding, respectively.
the affinity of BSA to the surface because the protein was
presumably attached onto the surface via hydrophobic interaction.
To form more complex cell patterns, we inserted a photomask
at the field diaphragm of the fluorescence microscope. In this
configuration, light from the mercury lamp was projected to the
sample in the same pattern as that of the inserted photomask (Figure
3a,c). The sizes of the irradiated regions were proportional to the
sizes of the spots on the photomasks and were inversely proportional
to the magnification of the objective lenses. On the basis of this
relationship, a cell array of 120-µm-square spots was formed by
using a photomask of an array pattern of 400-µm-square spots
(Figure 3b). A photomask of 100-µm-square spots gave 30-µm-
cell-adhesive spots, which is comparable to the size of single COS7
cells (Figure 3d). These results demonstrate that the present strategy
offers a simple method for forming cell-adhesive spots, and their
sizes can be reduced to the single cell level.
Figure 4. UV-directed positioning of single cells in proximity to cells
attached in advance. (a and b) Phase-contrast and fluorescence images of
substrates before and after seeding of fluorescent HEK293 cells where
unstained HEK293 cells (a) or COS7 cells (b) were attached in advance.
Second cells were stained with CellTracker Green CMFDA (Molecular
Probes). Yellow squares represent the irradiated regions. A fluorescence
image (red) is merged with the corresponding phase-contrast image (green).
Research Center Project from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan. J.N. acknowledges the
Special Postdoctoral Researcher Program of RIKEN.
The most important point is that the present strategy allows
formation of new cell-adhesive regions during cell cultivation. After
culturing unstained single HEK293 cells for 1 day, other regions
with a size comparable to that of single cells were irradiated in
proximity to the cells attached in advance (Figure 4a). The medium
was replaced with that containing fibronectin, and fluorescent
HEK293 cells were seeded. The fluorescent single cells selectively
attached to the irradiated regions (Figure 4a). To our knowledge,
this is the first demonstration of positioning of single cells in
proximity to cultivating single cells.
Moreover, two single cells of different types can be positioned
in a similar way. In Figure 4b, a single fluorescent HEK293 cell
was placed in proximity to a cultivating COS7 cell. Such positioning
of two single cells of different types is physiologically interesting
and will be useful for studying cell-cell interactions at the single
cell level.
In summary, we have developed a simple method for spatiotem-
poral control of cell adhesion on a substrate modified with a
photochemically active compound, whereon a protein preventing
cell adhesion is replaced with one promoting cell adhesion in
response to light. The present method makes it possible to form
various cell patterns using standard fluorescence microscopes.
Positioning of single cells in proximity to other single cells attached
in advance is also achieved. The sizes, shapes, and locations of the
cell-adhesive regions can be designed during the observation of
cultivating cells, and thus the substrate will be a useful dynamic
scaffold for studying cell-cell interactions.
Supporting Information Available: Experimental detail, contact
angle measurement, immunofluorescence study (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
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Acknowledgment. This work was supported, in part, by a
Grant-in-Aid for Young Scientists (B) (to J.N.), and the High-Tech
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