Photoactivation of a Substrate for Cell Adhesion under Standard Fluorescence Microscopes

Nakanishi, Jun; Kikuchi, Yukiko; Takarada, Tohru; Nakayama, Hidekazu; Yamaguchi, Kazuo; Maeda, Mizuo

doi:10.1021/ja044684c

Cited by 174 publications

(128 citation statements)

References 18 publications

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“…The substrate was placed upside down in a glass-bottom dish filled with water and irradiated using an inverted fluorescence microscope (IX81-PAFM, Olympus, Tokyo, Japan) through a stripe-patterned photomask inserted at the field diaphragm, as described previously [24,25]. After irradiation for 10 J cm −2 (e.g.…”

Section: Patterning His-tagged Proteins and Their Immunofluorescence mentioning

confidence: 99%

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

Nakanishi

Nakayama

Yamaguchi

et al. 2011

Science and Technology of Advanced Materials

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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) (EG 7 ). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni 2+ -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 EG 7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment ) 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 FNIII 7−10 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.

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Section: Patterning His-tagged Proteins and Their Immunofluorescence mentioning

confidence: 99%

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

Nakanishi

Nakayama

Yamaguchi

et al. 2011

Science and Technology of Advanced Materials

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“…2a). 19,67,68 "Caged" means "protected by a photocleavable protecting group". We have termed this dynamic substrate as a caged culture substrate.…”

Section: ·3 Substrates Responsive To Lightmentioning

confidence: 99%

Recent Advances in Cell Micropatterning Techniques for Bioanalytical and Biomedical Sciences

et al. 2008

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Cell micropatterning is a method for controlling the placement of living cells on a substrate surface. [1][2][3][4][5][6][7][8] It is important for a wide range of applications, such as tissue engineering, cellbased drug screening, and fundamental cell biology studies. Most cell micropatterning methods fall into three categories based on strategy: (1) seeding cells on a chemically patterned surface of different cell adhesiveness, (2) seeding cells on a topographically patterned surface, or (3) directed delivery of cells onto discrete regions of a substrate. Although the latter two categories are also important, this review mainly focuses on the first category. This is because it is the most common strategy and it provides tools to study fundamental aspects of cell adhesion at the single protein/receptor molecule level. 9There are excellent reviews that also cover the latter two categories. 3,8,10 We first introduce some of the important applications of cell micropatterning in general. We then describe methods for micropatterning the cell adhesiveness, referring to materials that promote or inhibit cell adhesion. We then move on to much more sophisticated substrates, so-called "dynamic substrates", whose cell adhesiveness can be changed by an external stimulus, such as heat, voltage and light. As an example of dynamic substrates, we describe a caged culture substrate developed by our group. This type of functional substrate opens up new possibilities in bioanalytical and biomedical sciences. Cell micropatterning is an important technique for a wide range of applications, such as tissue engineering, cell-based drug screening, and fundamental cell biology studies. This paper overviews cell patterning techniques based on chemically modified substrates with different degrees of cell adhesiveness. In particular, the focus is on dynamic substrates that change their cell adhesiveness in response to external stimuli, such as heat, voltage, and light. Such substrates allow researchers to achieve an in situ alteration of patterns of cell adhesiveness, which is useful for coculturing multiple cell types and analyzing dynamic cellular activities. As an example of dynamic substrates, we introduce a dynamic substrate based on a caged compound, where we accomplished a light-driven alteration of cell adhesiveness and the analysis of a single cell's motility.

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“…Such "in-situ"or "dynamic" patterning has been realized, for example, by using photoresponsive polymers [23,24] or laser ablation [25,26], just to name a few. We previously reported on the application of laser ablation phenomena to create growth pathways of neuronal processes (neurites) at sub-cellular resolution in situ [26].…”

Section: Introductionmentioning

confidence: 99%

Surface Modification of Cell Scaffold in Aqueous Solution Using TiO2 Photocatalysis and Linker Protein L2 for Patterning Primary Neurons

Sekine

Yamamoto

Kono

et al. 2015

e-J. Surf. Sci. Nanotech.

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Titanium dioxide (TiO2) photocatalysis can be applied to pattern proteins and cells under aqueous solution. In this work, we extended the application of this technique to patterning primary neurons, a type of cell with relatively weak adhesibility. For this purpose, we employed ribosomal protein L2 (RPL2) that has high affinity toward silica and metal oxides, including TiO2, to stably bind a neuronal adhesion protein laminin to the TiO2 surface. We utilized two types of molecular recognition to achieve this-binding of anti-laminin antibody to its antigen (laminin) and binding of protein A to the antibody. We show that a protein complex consisting of laminin/anti-laminin antibody/protein A-RPL2 is spontaneously formed by simply mixing the precursor proteins in solution phase. We then show that the surface coated with the protein complex supports stable growth of rat hippocampal neurons. Finally, we show that the cells can be selectively grown on the protein complex patterned with the TiO2-assisted method. The protocol established in this work is a unique combination of a top-down micropatterning of the surface using TiO2 photocatalysis and a bottom-up self-assembly of biomolecules, which can be further applied to pattern a wide range of proteins and cells.

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Photoactivation of a Substrate for Cell Adhesion under Standard Fluorescence Microscopes

Cited by 174 publications

References 18 publications

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

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

Recent Advances in Cell Micropatterning Techniques for Bioanalytical and Biomedical Sciences

Surface Modification of Cell Scaffold in Aqueous Solution Using TiO<sub>2 </sub>Photocatalysis and Linker Protein L2 for Patterning Primary Neurons<i><sup> </sup></i>

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