RSC_CC_C1CC15046D 1..3 - Biomedical Engineering

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Photolabile micropatterned surfaces for cell capture and releasew
Downloaded by University of California - Davis on 18 October 2011
Published on 05 October 2011 on http://pubs.rsc.org | doi:10.1039/C1CC15046D
Dong-Sik Shin,a Jeong Hyun Seo,a Julie L. Sutcliffeabc and Alexander Revzin*a
Received 13th August 2011, Accepted 20th September 2011
DOI: 10.1039/c1cc15046d
A method for capture and release of cells was developed using a
photolabile linker and antibody-attached glass surface with a
poly(ethylene glycol) (PEG)-pattern.
Surfaces are frequently micropatterned with proteins in order
to capture and culture cells in distinct geometric configurations.
Such micropatterned surfaces have important applications in
cell-based diagnostics, tissue engineering and developmental
biology.1 A variety of strategies for micropatterning cells on
surfaces have been described including soft lithography,2
microfluidics,3 optical tweezers,4 inkjet printing5 and photolithography.6 However, it is often desirable to retrieve cells
from micropatterns for more thorough downstream analysis.
The methods for cell retrieval reported so far remain limited
and may be broadly categorized into physicochemical,
electrochemical or light-based. Physicochemical approaches
include the use of shear7 or proteases to dislodge cells from
protein coated surfaces.8 Other groups developed peptide-coated
cell adhesive surfaces that could be rendered non-adhesive by
chemical stimulation.9 Electrochemical approaches on the
other hand may be used to locally stimulate cell release by
applying voltage to individually addressable electrodes.10
While this approach has demonstrated merit, the need to
culture cells on a conductive substrate and to fabricate electrode
arrays for local cell release makes it somewhat complicated.
Laser capture microdissection is one example of light-based
cell retrieval approaches.11 Another example, developed by
Albritton and colleagues employed ‘‘micropallets’’ for cell
cultivation and laser activation to release both cells and pallets
from the surface.12 However, this cell retrieval approach
requires an expensive piece of equipment, the laser, thus
limiting wide use.
We hypothesized that T-cells expressing CD4 antigen could
be captured inside the microwells and then released by
UV-induced cleavage of Ab anchors. Here we describe the
development of photolabile protein micropatterns for capture
a
Department of Biomedical Engineering, University of California,
Davis, 451 East Health Sciences Dr #2619, Davis, CA95616, USA.
E-mail: [email protected]; Fax: +1 530 754 5739;
Tel: +1 530 752 2383
b
Division of Hematology/Oncology, Department of Internal Medicine,
University of California, Davis, CA95616, USA
c
Center for Molecular and Genomic Imaging, University of
California, Davis, CA95616, USA
w Electronic supplementary information (ESI) available: Experimental
details and additional scheme and figures. See DOI: 10.1039/
c1cc15046d
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c
The Royal Society of Chemistry 2011
and light-triggered release of cells. In this approach, glass
surfaces were modified using a mixture of amine- and acrylateterminated methoxysilanes13 and then micropatterned using
poly(ethylene glycol) (PEG) photolithography14 (see electronic
supplementary information (ESI) for detailed descriptionw).
The mixed silane layer served a dual function: acryl groups
helped to anchor PEG gel microwells and amine groups
provided sites for covalent attachment of biomolecules within
the microwells. These surfaces were further functionalized
with azabenzotriazol-activated and 9-fluorenylmethoxycarbonyl
(Fmoc)-protected photolabile linker containing photosensitive
o-nitrobenzyl group (Scheme 1). Fmoc-groups were removed
and photolabile molecules were then reacted with biotin-NHS
(see ESIw for experimental detail). The micropatterned surfaces
were further functionalized with avidin and biotin-anti-CD4
antibodies (Abs). This multi-step surface modification protocol
resulted in formation of microwells with non-fouling walls and
Ab-containing cell capture sites anchored to glass via photocleavable molecules.
Prior to carrying out cell capture and release experiments,
we characterized release of avidin-biotin-photolabile linker
construct from the surface. Two types of experiments,
described diagrammatically in Scheme S1,w were carried out.
In the first set of experiments, photolabile linker and biotin
layers were assembled on amine/acryl functionalized glass,
forming a uniform biomolecular layer. This surface was then
exposed to UV light through a photomask so as to illuminate
majority of the surface except for circular 100-mm diameter
regions. Incubation of this surface with fluorescently-labeled
streptavidin revealed much stronger fluorescence emanating
from circular regions compared to background (B4 : 1 SNR,
See Fig. 1A and C). This experiment demonstrated cleavage of
biotin-photolabile linker constructs in the regions exposed to
UV and retention of biotin in the circular region protected
from UV light. In the data shown in Fig. 1A and C, surfaces
were exposed to 365-nm UV at 1.2 W cm 2 for 0.5 s.
In the second set of experiments, we sought to characterize
avidin detachment from photolabile surfaces. Direct exposure
of surfaces containing fluorescently-labeled avidin was unsuccessful
due to photobleaching. As an alternative strategy, mixedsilane containing surfaces were functionalized with photolabile
linker, followed by biotin and neutravidin as described before
(see Scheme S1w for details) and then exposed to 365-nm UV
at 1.2 W cm 2 for 0.5 s through photomask containing 100-mm
diameter darkfield regions. This treatment was expected to
result in cleavage of the neutravidin-terminated biomolecular
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Fig. 1 Avidin patterning by treating (A) Alexa Fluor 546-streptavidin
after photolithography of surface bound biotin and (B) Atto
565-Biotin after photolithography of surface bound neutravidin; (C)
fluorescence profile of the white line in (A); (D) fluorescence profile of
the white line in (B).
Scheme 1 Surface modification and cell attachment/detachment on
PEG patterned surface with photolabile linkage: (a) amino- and acrylmixed silanization, (b) selective polymerization of PEG by using
photolithography, (c) coupling of photolabile linker, (d) coupling of
biotin, (e) attachment of avidin and subsequent attachment of biotinanti-CD4 Ab, (f) capture of cells, (g) release of cells.
layer. Incubation of this surface with fluorescently-labeled
biotin revealed much stronger fluorescence signal in circular
regions that were not illuminated by UV and were expected to
retain neutravidin molecules (see Fig. 1B and D). However,
the ratio of on the spot vs. off the spot fluorescence was 2 : 1
(Fig. 1D), suggesting some neutravidin molecules in the
regions exposed to UV were still retained on the surface.
In the future, molecules such as PEG or chitosan may be
incorporated into the biointerface design to further minimize
non-specific binding and to facilitated more efficient protein
release upon UV activation.15
In addition to micropatterning and fluorescence imaging,
ellipsometry was employed to monitor the photocleavage of
biomolecular constructs. These experiments were performed
on silicon wafer pieces functinalized in the manner identical to
that described for glass. Fig. 2A shows ellipsometry measurements performed at each step of the surface modification
protocol. These results show a 7.2-nm thickness increased
Chem. Commun.
Fig. 2 (A) Average surface thickness from ellipsometry scanning the
surface: (a) Oxygen plasma treated surface; (b) amino- and acrylmixed silane surface; (c) photolabile linker and biotin coupled surface;
(d) avidin attached surface; (e) UV exposed surface on (d). (B) Degree
of neutravidin detachment by UV exposure.
after silanization, suggesting presence of a multi-layer silane
film.16 Assembly of photolabile linker and biotin caused a
further thickness increase of 1.4 nm, while attachment of
neutravidin added another 3.0 nm to the multi-layer construct.
The overall thickness of this photolabile biointerface was 11.5 nm
before and 10.3 nm after UV exposure. This experiment points
to partial removal of molecular from photolabile interface
upon UV exposure. Plot of thickness change vs. time of
UV exposure shown in Fig. 2B demonstrates that no appreciable
cleavage was observed after 15 min by UV exposure at
1.8 mW cm 2 (Fig. 2B).
To prove the concept of UV triggered cell release, PEG
microwell arrays containing photolabile avidin terminated
attachment regions were functionalized with anti-CD4-biotin.
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References
Fig. 3 Cell populations in wells before and after UV exposure: (A)
before UV exposure; (B) at 2 min UV exposure; (C) count of residingin cells depending on UV exposure.
These microwells were then incubated with CD4 antigen
expressing T-cells (Molt-3). Fig. 3A shows selective attachment
of cells inside the microwells with minimal attachment occurring
on non-fouling PEG hydrogel regions. Molt-3 cells are nonadherent CD4 expressing lymphoblasts that were captured inside
the microwells solely due to the presence of anti-CD4 Ab. They
did not spread on the surface but they were immobilized on the
surface with antibody-cell interaction. Hydrogel microwells
remained adherent on the surface for 5 to 7 days. Glass slides
with cell arrays were placed in a Petri dish and exposed to 365-nm
UV at 500 mW cm 2. Cells started detaching immediately
upon UV exposure and 90% of T-cells were detached after 1
min exposure followed by gentle rinsing (Fig. 3B and C).
Importantly, Fig. S2w demonstrates that conditions used for
cell release did not adversely affect cell viability.
In conclusion, we have developed micropatterned photolabile
surfaces for capture and light-triggered release of cells. These
surfaces consisted of microwell arrays with non-fouling PEG
gel walls and cell adhesive photolabile glass bottom. Cleavage
of biointerface components was characterized by fluorescence
microscopy and ellipsometry. As a proof of concept demonstration, microwell arrays were functionalized with anti-CD4
Ab and were used to capture CD4-expressing T-cells (Molt-3
cells). A short exposure to UV was sufficient to cleave anchors
holding cells to the surface, leading to cell release upon gentle
agitation. Importantly, this cell retrieval protocol did not
compromise cell viability. The biointerface design described
here is an important addition to a limited repertoire of cell retrieval
technologies reported in the literature. Unlike other lighttriggered release technologies based on lasers, this approach
utilizes standard UV sources available in most laboratories. In
the future, we envision integrating this cell retrieval approach
with single cell detection strategies under development in our
lab13,17 to enable release of specific groups of cells based on
secreted signalling molecules.
This work was supported by NIH grant RC4EB012836-01
awarded to JS and NSF EFRI grant awarded to AR.
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