Switchable adhesive substrates: revealing geometry dependence in collective cell behavior.

Collective cell migration plays a major role in cancer metastasis and wound healing, therefore, several in vitro assays for studying such behavior have been developed. Using photoswitchable surfaces, we studied collective cell expansion behavior from initially precisely controlled adhesive patterns. A non-adhesive poly(ethylene glycol) (PEG) layer is conjugated to a glass coverslip via 2-nitrobenzyl groups, which cleave upon exposure to UV light, changing the surface from non-cell-adhesive to cell-adhesive without mechanical interference. Initial cell attaching areas are generated in arbitrary shapes via projection exposure through a photomask. After a growth phase, epithelial cell sheets are released from their confinement by a second illumination allowing for collective cell expansion. Our experiments with epithelial cells show that cluster size and boundary curvature modulate the expansion of the cell sheet and the formation of leader cells. At a certain cluster size, characteristics of the expansion behavior change and cells in the core are hardly affected by the boundary release. With donut-like ring structures, we demonstrate a break in symmetry between the behavior of cells along the outer convex boundary and along the inner concave boundary. Additionally, we observe that collective migration characteristics are modulated by the initial incubation time of the cell sheet.

Photocontrol of cell adhesion on amino-bearing surfaces by reversible conjugation of poly(ethylene glycol) via a photocleavable linker.

Dynamic control of cell adhesion on substrates is a useful technology in tissue engineering and basic biology. This paper describes a method for the control of cell adhesion on amino-bearing surfaces by reversible conjugation of an anti-fouling polymer, poly(ethylene glycol) (PEG), via a newly developed photocleavable linker, 1-(5-methoxy-2-nitro-4-prop-2-ynyloxyphenyl)ethyl N-succinimidyl carbonate (1). This molecule has alkyne and succinimidyl carbonate at each end, which are connected by photocleavable 2-nitrobenzyl ester. Under this molecular design, the molecule crosslinked azides and amines, whose linkage cleaved upon application of near-UV light. By using aminosilanised glass and silicon as model substrates, we studied their reversible surface modification with PEG-azide (M(w) = 5000) based on contact angle measurements, ellipsometry, and AFM morphological observations. Protein adsorption and cell adhesion dramatically changed by PEGylation and the following irradiation, which can be used for cellular patterning. Also, the capability of the substrate to change cell adhesiveness by photoirradiation during cell cultivation was demonstrated by inducing cell migration. We believe this method will be useful for dynamic patterning of cells on protein-based scaffolds.

Dynamic culture substrate that captures a specific extracellular matrix protein in response to light.

The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG7). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni2+-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII7-10) to the irradiated regions. In contrast, when bovine serum albumin-a major serum protein-was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

Silane coupling agent bearing a photoremovable succinimidyl carbonate for patterning amines on glass and silicon surfaces with controlled surface densities.

Patterned immobilization of synthetic and biological ligands on material surfaces with controlled surface densities is important for various bioanalytical and cell biological purposes. This paper describes the synthesis, characterization, and application of a novel silane coupling agent bearing a photoremovable succinimidyl carbonate, which enables the photopatterning of various primary amines on glass and silicon surfaces. The silane coupling agent is 1-[5-methoxy-2-nitro-4-(3-trimethoxysilylpropyloxy)phenyl]ethyl N-succinimidyl carbonate. The distinct feature of this molecule is that it has a photocleavable 2-nitrobenzyl switch between a trimethoxysilyl group and a succinimidyl carbonate, each reactive to the hydroxy groups of inorganic oxides and primary amines. Based on this molecular design, the compound allows for the one-step introduction of succinimidyl carbonates onto the surface of glass and silicon, immobilization of primary amines, and region-selective and dose-dependent release of the amines by near-UV irradiation. Therefore, we were able to pattern amine ligands on the substrates in given surface densities and arbitrary geometries by controlling the doses and regions of photoirradiation. These features were verified by UV-vis spectroscopy, contact angle measurements, infrared (IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), ellipsometry, and atomic force microscopy (AFM). The compound was applied to form a chemical density gradient of amino-biotin on a silicon substrate in a range of 0.87-0.12 chains/nm(2) by controlling photoirradiation under a standard fluorescence microscope. Furthermore, we also succeeded in forming a chemical density gradient at a lower surface density range (0.15-0.011 chains/nm(2)) on the substrate by diluting the feed amino-biotin with an inert control amine.

Light-regulated activation of cellular signaling by gold nanoparticles that capture and release amines.

A photoresponsive nanocarrier for amines based on gold nanoparticles (GNPs) having a photocleavable succinimidyl ester has been developed. It offers a useful platform for the synthesis of caged compounds. Using the GNPs, we have developed caged histamine for the first time and applied it to evoke intracellular signaling by controlled near-UV irradiation. The present work will allow for new possibilities in studies of inter- and intracellular signaling networks.

Arraying heterotypic single cells on photoactivatable cell-culturing substrates.

This article describes a photochemical method for the site-selective assembly of heterotypic cells on a glass substrate modified with a silane coupling agent having a caged functional group. Silane coupling agents having a carboxyl (COOH), amino (NH 2), hydroxyl (OH), or thiol (SH) group protected by a photocleavable 2-nitrobenzyl group were synthesized to modify the surfaces of glass coverslips. The caged substrates were first coated by the adsorption of a blocking agent, bovine serum albumin (BSA), to make the entire surface non-cell-adhesive and then irradiated at 365 nm under a standard fluorescence microscope. The photocleavage reaction on the surface was followed by contact angle measurements and X-ray photoelectron spectroscopy. When COS7, NIH3T3, and HEK293 cells were seeded onto these substrates in a serum-free medium, the cells adhered selectively and efficiently to the irradiated regions on the caged NH 2 substrate, whereas the other caged COOH, SH, and OH substrates were nonphotoactivatable for cell adhesion. Qualitative and quantitative analysis of BSA adsorbed to the uncaged substrates revealed that this highly efficient photoactivation on the caged NH 2 substrate arose because of the following reasons: (i) upon photoactivation, BSA adsorbed in advance on the 2-nitrobenzyl groups was readsorbed onto the uncaged functional groups and (ii) BSA readsorbed onto the NH 2 groups became unable to passivate the surface against cell adhesion whereas BSA on the other groups still had normal passivating activity. It was also demonstrated that heterotypic single COS7, NIH3T3, and HEK293 cells were positioned at any desired arrangement on the caged NH 2 substrate by repeating the UV irradiation at optimized array spot sizes and cell seeding in optimized cell concentrations. The present method will be particularly useful in studying the dynamic processes of cell-cell interactions at a single-cell level.

Spatiotemporal control of cell adhesion on a self-assembled monolayer having a photocleavable protecting group.

Control of cell adhesion is a key technology for cell-based drug screening and for analyses of cellular processes. We developed a method to spatiotemporally control cell adhesion using a photochemical reaction. We prepared a cell-culturing substrate by modifying the surface of a glass coverslip with a self-assembled monolayer of an alkylsiloxane having a photocleavable 2-nitrobenzyl group. Bovine serum albumin (BSA) was adsorbed onto the substrate to make the surface inert to cell adhesion. When exposed to UV light, the alkylsiloxane underwent a photocleavage reaction, leading to the release of BSA from the surface. Fibronectin, a protein promoting cell adhesion, was added to cover the irradiated regions and made them cell-adhesive. Seeding of cells on this substrate resulted in their selective adhesion to the illuminated regions. By controlling the sizes of the illuminated regions, we formed cell-adhesive spots smaller than single cells and located focal adhesions of the cells. Moreover, by subsequently illuminating the region alongside the cells patterned on the substrate in advance, we released their geometrical confinements and induced migration and proliferation. These manipulations were conducted under a conventional fluorescence microscope without any additional instruments. The present method of cell manipulation will be useful for cell biological studies as well as for the formation of cell arrays.

Photoactivation of a substrate for cell adhesion under standard fluorescence microscopes.

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 photoactivation of a substrate for cell adhesion under standard fluorescence microscopes. A glass substrate chemically modified with an alkylsiloxane having a photocleavable 2-nitrobenzyl group was coated with bovine serum albumin to prevent cell adhesion. Upon irradiation under a fluorescence microscope, the protein was replaced with fibronectin, which made the irradiated region cell-adhesive. Subsequent seeding of HEK293 or COS7 cells produced patterns corresponding to the irradiated patterns. We succeeded for the first time in positioning single cells in proximity to cultivating single cells. The present method provides a general strategy for positioning single cells of same or different types at any locations on the substrate and will be useful for studying cell-cell interactions.