Authors thank Mr. Kotaro Okubo for the kind assistance with SEM imaging. This work was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Basic Research (B) (20310069), Grant-in-Aid for Research Activity Start-up (25880021), and by research grants from the Kurata Memorial Hitachi Science and Technology Foundation and the Nippon Sheet Glass Foundation for Materials Science and Engineering.

1. Preparation of TiO2-coated Glass Coverslip
<ol>
	<li>Number the coverslips using a diamond scriber. This helps not only to keep track of each coverslips but also to ensure that the correct side of the sample is facing up. Clean the coverslips, first under running ddH2O, then by immersing them in piranha solution (H2SO4:H2O2 = 4:1). After 10 min, rinse the coverslips thoroughly, 8 times in ddH2O. Dry the coverslips under N2 flow.<...

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In our current protocol, TiO2 film was formed by RF-magnetron sputtering. We favor this method of deposition since it allows us to reproducibly prepare a photocatalytic TiO2 film with a sub-nm roughness. Although sputter deposition processes are familiar to materials scientists and electronic engineers, it may not be quite accessible to biologists. In that case, spin-coated TiO2 film would be an alternative choice23. In this method, TiO2 nanoparticles dissolved in a ...

Semiconductor lithography processes and its derivatives &#8212; such as photolithography1,2, electron-beam lithography3-6, and microcontact printing7-10 &#8212; have now become an established tool in cell biology to grow living cells in a defined position and geometry. The patterning method relies on the use of microfabricated substrates, consisting of micro-island of cell permissive coating in a non-permissive background. Such substrate serves as a template to pattern the cells. These technologies have provided us the novel methods to engineer cells and their function at a single- and multi-cellular level, to extract the intrinsic properties of cells, and to increase the throughput of cell-based drug screening11.
The degree-of-freedom in cell patterning would greatly increase if the template pattern geometry could be altered in situ, i.e., while cells are cultured on the surface. The conventional methods for pattern formation cannot be directly applied here, since they process samples in atmosphere or in vacuum. Therefore various new surface modification techniques have been proposed, which are based, e.g., on photoreactive compounds12,13 or laser ablation5,14, just to name a few. The proposed methods have been nicely reviewed by Robertus et al.15, and more recently by Choi et al.16 and by Nakanishi17.
Here in this article, we describe a novel protocol of in-situ surface modification, which takes advantage of photocatalytic decomposition of organic molecules on a titanium dioxide (TiO2) surface18,19. In this method, a TiO2 film is inserted between the glass substrate and the organic film that interfaces the cells, and the organic film is decomposed in situ by locally irradiating ultraviolet (UV) light to a region of interest (&#955; &#60; 388 nm). We show that the new protocol can be used to create micropatterns of extracellular matrix proteins and living cells both ex situ and in situ. TiO2 is biocompatible, chemically stable, and optically transparent, features of which makes it friendly to introduce in cell-culture experiments. This protocol provides a materials science-based alternative for modifying cell-culture scaffolds in cell-culture environment.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass coverslip</td>
			<td>Warner Instruments</td>
			<td>CS-15R15</td>
			<td>15 mm, #1.5 thickness</td>
		</tr>
		<tr>
			<td>Diamond scriber</td>
			<td>Ogura Jewel Industry</td>
			<td>D-Point Pen</td>
			<td></td>
		</tr>
		<tr>
			<td>RF sputtering system</td>
			<td>ANELVA</td>
			<td>SPC350</td>
			<td></td>
		</tr>
		<tr>
			<td>TiO2 sputtering target</td>
			<td>Kojundo Chemical Lab</td>
			<td>Titanium (IV) oxide, target</td>
			<td>Purity, 99.9%</td>
		</tr>
		<tr>
			<td>Plasma reactor</td>
			<td>Yamato</td>
			<td>PR301</td>
			<td></td>
		</tr>
		<tr>
			<td>n-octadecyltrichlorosilane 
			(OTS)</td>
			<td>Aldrich</td>
			<td>104817</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Wako</td>
			<td>204-01866</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture dish (35 mm)</td>
			<td>Greiner</td>
			<td>627160</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture dish (60 mm)</td>
			<td>BD Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Type-IV collagen</td>
			<td>Nitta Gelatin</td>
			<td>Cellmatrix Type IV</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td>Heat-inactivate and pass through a 0.22 mm filter before use</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td>Pass through a 0.22 mm filter before use</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (100x)</td>
			<td>Nacalai tesque</td>
			<td>26253-84</td>
			<td></td>
		</tr>
		<tr>
			<td>7S nerve growth factor (NGF)</td>
			<td>Alomone Labs</td>
			<td>N-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Dojindo</td>
			<td>N001</td>
			<td>Stock solution in 0.5 M</td>
		</tr>
		<tr>
			<td>TiO2 nanoparticle</td>
			<td>Tayca</td>
			<td>TKD-701</td>
			<td></td>
		</tr>
	</tbody>
</table>

photopatterning proteins and cells in aqueous environment using tio2 photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO2 photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO2 film was sputter-coated on a glass coverslip, and the TiO2 surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.

Figure 2A shows a cross-sectional scanning electron microscopy (SEM) image of the sputter-deposited TiO2 film. From the observation, thickness of the film was estimated to be approximately 150 nm. Noticeable here is the flatness of the deposited TiO2 film. Further analysis by atomic force microscopy (AFM) revealed that the root-mean-square (rms) roughness of the surface was 0.2 nm (Figure 2B).
When the TiO2 surface is modified ...

Watch this Scientific Journal Video about Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis at JoVE.com

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

photopatterning-proteins-cells-aqueous-environment-using-tio2

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Bioengineering

7.8K visualizações.  Tohoku University. We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.