Surface Modification of Cell Scaffold in Aqueous Solution Using TiO₂Photocatalysis and Linker Protein L2 for Patterning Primary Neurons<i></i>

Abstract

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.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO₂ Photocatalysis

“…Thus far, we have utilized this method in patterning not only the PC12 cells but also a human pancreatic cancer cell line AsPC-1 and primary neurons obtained from embryonic rat hippocampi 19 . Adhesibility of AsPC-1 cells are much stronger than that of PC12 cells or hippocampal neurons, and supplementation of scaffolding molecules, such as collagen, was not required in patterning them.…”

“…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 (TiO 2 ) surface 18,19 . In this method, a TiO 2 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 (λ < 388 nm).…”

“…We tried other surface coatings, such as polyethylene glycol-silane SAMs and perfluoro-silane SAMs, to find that none of these were anti-fouling to poly-lysine. To solve this issue, we very recently showed that a cross-linking molecule could be used to induce active adsorption of the scaffolding molecules onto the UV-irradiated region 19 . Such selection of scaffolding molecules and optimization of its concentration should be carried out for each experiment.…”

In situ modification of cell-culture scaffolds by photocatalysis of visible-light-responsive TiO2film

“…Note that the TiO 2 film sputter-deposited at room temperature without O 2 gas injection is the film we used in our previous work. [19][20][21] This indicates that the TiO 2 film exhibits photocatalytic activity upon UV exposure, although the film does not have a distinct crystal structure. If crystallinity is not always required for the photocatalytic activity of TiO 2 , the film is expected to have the VLresponsive activity since the film exhibits the absorbance in the VL region.…”

“…One promising method for the in situ cell-culture scaffold modification is TiO 2 photocatalysis, as reported previously. [19][20][21] A transparent and chemically stable TiO 2 film is compatible with conventional cell-culture experiments. Focused ultraviolet (UV) light or scanning UV laser makes it possible to decompose surface molecules in the medium in a region-selective manner.…”