Photocontrollable dynamic micropatterning of non-adherent mammalian cells using a photocleavable poly(ethylene glycol) lipid

Article abstract of DOI:10.1002/anie.201106106

Cells in the spotlight: A substrate was coated with a poly(ethylene glycol) (PEG) segment bound to a lipid through a linker that was cleaved by UV irradiation to leave a PEG-coated area. Irradiation of an arbitrary pattern easily and rapidly yielded a hig

Full text of DOI:10.1002/anie.201106106

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Angewandte
Communications
DOI: 10.1002/anie.201106106
Cell Patterning
Photocontrollable Dynamic Micropatterning of Non-adherent
Mammalian Cells Using a Photocleavable Poly(ethylene glycol)
Lipid**
Satoshi Yamaguchi,* Shinya Yamahira, Kyoko Kikuchi, Kimio Sumaru, Toshiyuki Kanamori,
and Teruyuki Nagamune*
Cell micropatterning has become an important technology for
a wide variety of applications, ranging from tissue engineer-
ing[1] and cell microarrays[2] to fundamental studies in cell
biology.[3] In addition to conventional patterning methods,
such as photolithography, soft lithography, and inkjet printing,
patterning methods with dynamic substrates, in which cell
adhesive properties can be changed by an external stimulus,
such as heat,[4] voltage,[5] and light,[6] at any desired position
and any point in time, are currently the focus of many studies.
These spatiotemporal patterning methods can easily construct
patterns of multiple cell lines and be useful for analyzing
dynamic cellular activity.[7] In particular, in contrast to heat
and voltage, light can be readily applied to anywhere in
transmissive spaces with high spatial and temporal resolution,
and light-induced fine control of biomolecules or living cells
has been widely reported,[6,8] even at a single-molecular
level.[8b] Therefore, cell patterning with light-responsive
substrates potentially offers a practical tool for biologists.
However, almost all reported cell micropatterning methods
have a major limitation in target cells. In conventional
methods, the adhesiveness of cells is used to attach them
onto bare or ligand-coated surfaces. Therefore, the existing
methods cannot be applied to non- or weakly adherent cells,
which include blood cells (especially immunocytes), some
cancer cells, and stem cells. These cell lines are important as
research targets in biological and medical fields, and for this
reason expansion of an applicable range of current micro-
patterning methods to non-adherent cells is an important
challenge.
We report herein a light-induced in situ cell micropattern-
ing method that can be applied to non-adherent cells.
Recently, we reported a cell patterning method for non-
adherent cells using a cell membrane binding reagent
consisting of poly(ethylene glycol) (PEG) and an oleyl
group.[9] This compound can bind to any type of cell without
cytotoxicity, because the oleyl moiety can be inserted into
ubiquitous lipid bilayer membranes in a noncovalent man-
ner.[9a] In the current study, a photocleavable PEG-lipid was
newly designed and synthesized for light-induced cell pat-
terning, and then cell immobilization on the substrate coated
with the photocleavable PEG-lipid was confirmed to be
regulated by the dose of light exposure. Moreover, the present
method allows the preparation of arbitrary and fine patterns
of non-adherent cells. Furthermore, the cell micropattern on
the present light-responsive substrate can be altered by light
irradiation at a desired point in time.
First, we designed and synthesized a photocleavable PEG-
lipid. In our design, a photocleavable unit was incorporated
between the PEG and oleyl moieties, and at the opposite end
of the PEG segment an amino-reactive ester group was added
for attachment onto the substrate through an amide coupling
reaction (Figure 1a). After coating, the oleyl moieties are
expected to be exposed and to anchor living cells (Figure 1b).
Moreover, this molecule can be cleaved by irradiation, and
then the PEG moiety is exposed at the light-irradiated area
(Figure 1b). It has been reported that a PEG-coated surface
inhibits cell adhesion.[10] Therefore, cell-adhesive and non-
adhesive surfaces were expected to be prepared by light
irradiation (Figure 1b). A photocleavable PEG-lipid was
synthesized from a commercially available o-nitrobenzyl
photocleavable linker[11] and characterized by using standard
methods (see the Supporting Information). The photolytic
property of the PEG-lipid in solution was confirmed by means
[*] Dr. S. Yamaguchi, Prof. T. Nagamune
Department of Chemistry and Biotechnology
School of Engineering, The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan)
E-mail: yamaguchi@bio.t.u-tokyo.ac.jp
nagamune@bioeng.t.u-tokyo.ac.jp
S. Yamahira, Prof. T. Nagamune
Department of Bioengineering
School of Engineering, The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan)
1
of H NMR spectroscopy after irradiation with UV light at
365 nm (see the Supporting Information). Furthermore,
substrate coating by the photocleavable PEG-lipid and its
photolytic degradation on substrates were confirmed by
water-drop contact-angle measurements (see the Supporting
Information).
Cell immobilization on a dish coated with photocleavable
PEG-lipid was investigated by fluorescent microscopic obser-
vation before and after irradiating the dish with UV light
(365 nm). On the nonirradiated dishes, a non-adherent cell
line, the human hematopoietic cell line BaF3, was densely
K. Kikuchi, K. Sumaru, T. Kanamori
Research Center for Stem Cell Engineering
National Institute of Advanced Industrial Science and Technology
Tsukuba Central 5th, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565
(Japan)
[**] This work was supported by the Center for NanoBio Integration in
The University of Tokyo.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201106106.
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Figure 1. Schematic diagram of light-controllable cell micropatterning
and recovery on a substrate coated with photocleavable PEG-lipid.
a) The chemical structure of a photocleavable PEG-lipid consisting of
an oleyl group (red), an o-nitrobenzyl photocleavable linker (blue), a
PEG chain (green), and an amino-reactive N-hydroxysuccinimide ester
group (black). b) The substrate is coated with bovine serum albumine
(BSA) and the photocleavable PEG-lipid, and then the PEG moiety is
exposed to light in the unmasked area. When the cell suspension is
placed on this substrate and excess rinsed away, living cells are
patterned on the nonirradiated area through an interaction between
cell membranes and the exposed oleyl groups. The immobilized cells
are selectively recovered by light irradiation.
Figure 2. Light-induced inhibition of cell immobilization on a substrate
coated with photocleavable PEG-lipid. Fluorescent micrographs of
immobilized living cells on a substrate irradiated with UV light of a) 0,
b) 0.3, c) 0.6, and d) 1.0 JcmÀ2. Enhanced green fluorescent protein
(EGFP)-expressing BaF3 cells were used in this experiment. The scale
bar represents 100 mm. e) Relationship between the density of immo-
bilized cells and the light dose.
microscope.[12] Figure 3a shows the microscopic image of the
irradiated pattern. A highly contrasted cell pattern, in which
cells were immobilized only on the nonirradiated region, was
obtained as expected (Figure 3b). This result strongly indi-
cated that cells were firmly captured on the nonirradiated
region, which was attributed to interaction between cell
membranes and the oleyl moiety of the photocleavable PEG-
lipid. On the irradiated region, where the oleyl group was
removed through the cleavage of a nitrobenzyl group, cells
did not interact or weakly interacted with the exposed PEG
moiety and then they were washed off by rinsing. The present
finding of a high-contrast cell pattern is thought to be derived
from an adequate difference between the strength of the
interaction of the cell surface with the nonirradiated and
irradiated surfaces.
To evaluate the precision limit of the present patterning
method, we attempted to prepare cell arrays with various
sizes of cell spots (Figure 3c,d,e). The substrates were
irradiated with inverted dot patterns of light with various
dot diameters (100, 50, and 25 mm). As a result, cell arrays
consisting of uniformly sized spots were obtained correspond-
ing to the diameters of the irradiation pattern, and the
precision of a single cell size was achieved by irradiating with
the finest pattern of light (Figure 3e). Consequently, by high-
resolution irradiation an extremely precise pattern of non-
adherent cells can be prepared by the present patterning
method.
immobilized by placing a cell suspension (6.1 ꢀ 106 cellsmLÀ1
)
on the surface for 15 min and rinsing with PBS (phosphate-
buffered saline, Figure 2a). On the irradiated dishes the
density of immobilized cells decreased corresponding to the
dose increase of UV light (Figure 2b,c,d). To demonstrate
the versatility of the present method, other non-adherent or
adherent cells, such as the human leukemic cell line K562 and
cervical carcinoma cell line HeLa, were immobilized on the
same substrates, and we found that the cell densities similarly
depended on the light dose. Figure 2e shows the relationship
between the density of immobilized cells and the light dose in
detail. A light dose above 0.8 J cmÀ2 was required to inhibit
cell immobilization. This result indicates that cell immobili-
zation can be controlled by irradiating the PEG-lipid-coated
substrate with a low dose of light. Furthermore, almost all
immobilized cells (98%) were alive on the present substrate
after incubation for one day, and cell growth was also
confirmed (see the Supporting Information). This result
indicates that the PEG-lipid-coated substrate is biocompat-
ible.
On the present substrates coated with photocleavable
PEG-lipid, cell patterns were successfully obtained by
irradiating with UV light (1 JcmÀ2) in an arbitrary pattern
before exposing the substrate to a cell suspension. We
employed a light-irradiation system based on a computer-
controllable microprojection unit in an optical system using a
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Figure 3. Micrographs of cell patterns on a substrate coated with
photocleavable PEG-lipid. a) Micrograph of the substrate from irradiat-
ing a pattern with UV light (365 nm). At the irradiated area of the
projected pattern, the light was reflected on the substrate and detected
as a blue pattern (light area). b) A phase-contrast micrograph of the
cell pattern on the substrate irradiated as shown in (a). c–e) Phase-
contrast micrographs of a cell microarray on a substrate irradiated by
inverted dot patterns of UV light (365 nm) with a diameter of c) 100,
d) 50, and e) 25 mm.
Figure 4. Light-induced cell detachment from a cell microarray on a
substrate coated with photocleavable PEG-lipid. a) Schematic illustra-
tion of light-induced cell detachment in a microfluidic device. b) A
phase-contrast micrograph of the microarray of BaF3 cells before and
c) after light-irradiation at 5.2 JcmÀ2. The linear flow rate was
20 cmsÀ1
.
Finally, to demonstrate that the immobilized cell pattern
can be spatiotemporally altered by light irradiation, we
attempted to detach immobilized cells from a targeted area
by light irradiation. The fluid shear stress on irradiated cells,
which was generated by rinsing treatment after light irradi-
ation, is critical for the detachment of cells from the
substrate.[13] Therefore, to precisely control the shear stress,
the cell pattern was constructed on a microchannel in a
microfluidic device, and the immobilized cells were irradiated
under constant shear stress by microfluidic control (Fig-
ure 4a). A cell microarray with a spot size of approximately
100 mm was prepared on the PEG-lipid-coated microchannel
by irradiation with an inverted dot pattern of light and
subsequent introduction of cell suspension into the micro-
channel. At a linear flow rate of 20 cmsÀ1 we found that the
cells were tightly immobilized on the nonirradiated dot spots,
whereas no cells remained on the irradiated area (Figure 4b).
At the same flow rate, a target cell spot was then continuously
irradiated with a spot of light (365 nm, 84 mWcmÀ2) until all
cells in the target spot were detached. During light irradi-
ation, the cells on the irradiated spot were detached one by
one and swept away in the flow (see Movie S1 in the
Supporting Information). After irradiation at 5.2 JcmÀ2, all
cells at the irradiated spot disappeared; however, almost all
cells remained at the nonirradiated spots (Figure 4c). This
light-irradiation experiment was also performed at 20 other
spots on the same microchannel or other microchannels. We
observed that all cells in the irradiated spots could be
reproducibly detached by light irradiation below 3.2 JcmÀ2,
while 94.8% of the immobilized cells at the nonirradiated
spots (n = 69) remained. Therefore, the immobilized cells
were selectively detached only from the irradiated area. We
assessed the viability of the photoirradiated-detached cells by
trypan blue exclusion. The viability of photoirradiated cells
((90 Æ 6.4) %) was almost the same as that of cells recovered
from a nonirradiated surface ((90 Æ 6.7) %; see the Support-
ing Information). These results show that, in principle,
immobilized cells can be selectively collected from the
present substrate by photoirradiation without cell damage.
Cell-sorting technology is a necessary tool for screening
and purifying target cells. However, in existing methods based
on flow cytometry, cells are evaluated only by the intensity of
fluorescence derived from a probe or marker gene, and
therefore false positive or negative cells are often sorted
because of their background fluorescence. Recently, a single-
cell microarray has been reported to enable high-throughput
screening of target cells with a higher accuracy, because the
same single cell can be analyzed before and after stimulation
or staining to cancel the background noise in probe signals.[14]
Moreover, on a single-cell microarray, high-content screening
can be performed by observing the changes in cell morphol-
ogy and cellular localization of specific proteins; such changes
cannot be detected by a flow cytometer. Although further
optimization of cell patterning and recovery conditions is
required, the present method using a photocleavable PEG-
lipid is a useful and promising tool for screening and retrieval
of target cells at the single-cell level. Furthermore, in
principle, the present micropatterning technology can be
applied to liposomes, exosomes, bacteria, and others that are
coated with a lipid bilayer membrane.
In summary, we have developed a light-controllable
micropatterning method for non-adherent cells by using a
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[4] M. Yamato, C. Konno, M. Utsumi, A. Kikuchi, T. Okano,
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coated with a photocleavable PEG-lipid could be controlled
by the dose of UV light and perfectly inhibited above
0.8 JcmÀ2. On the present photocontrollable substrates, a
highly contrasted cell pattern with the precision of a single
cell size was easily and rapidly obtained without cell damage
by irradiating an arbitrary pattern using a computer-control-
lable light-irradiation system. Furthermore, light-induced cell
detachment from the substrate was successfully accom-
plished, and this result indicated that target non-adherent
cells can be selectively recovered by light irradiation. There-
fore, the present cell patterning method is promising for
applications to fundamental studies of non-adherent cells at
the single-cell level and to cell screening and sorting
technologies.
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Keywords: cell adhesion · cell patterning · microarrays ·
.
microfluids · photolysis
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