光图案蛋白质和细胞中的水环境中使用二氧化钛<sub> 2</sub>光催化
Summary
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.
Abstract
吸附的二氧化钛（TiO 2的）的表面上的有机污染物可以通过光催化下的紫外线（UV）光分解。在这里，我们描述了一种新的协议采用二氧化钛光催化局部改变衬底表面的细胞亲和力。在这个实验中，一个薄的TiO 2膜为溅射镀膜在玻璃盖玻片，并在TiO 2表面随后改性以从十八烷基三氯硅烷（OTS）衍生的有机硅烷单层，其抑制细胞粘附。将样品浸在细胞培养基中，和聚焦UV光照射至一个八角形区域。当神经元细胞系PC12细胞铺在样本，细胞粘附只在紫外线照射的区域。进一步的研究表明这种表面改性也可以在原位进行，也就是说，即使当细胞生长在衬底上。适当的修改表面的所需的细胞外矩阵Protein胶原存在于在紫外线照射时的介质。这里提出的技术可以潜在地在图案形成多种细胞类型可以采用用于构建共培养系统或任意地操纵细胞培养下。
Introduction
半导体光刻工艺及其衍生物-诸如光刻1,2，电子束光刻3-6，和微接触印刷7-10 -现在已成为一个既定的工具在细胞生物学生长的活细胞在限定位置和几何形状。图案形成方法依赖于使用微制造基底的，由微岛细胞容许涂料在非允许的背景。例如基底用作模板图案的细胞。这些技术为我们提供了新的方法来设计的细胞和它们的功能在一个单和多细胞水平上，以提取细胞的固有特性，并增加细胞为基础的药物筛选11的吞吐量。
在细胞图案化的自由度度会大大增加，如果模板图案几何可以改变在原位 ， 即 ，当细胞是上一次一培养urface。常规方法的图案形成，不能直接应用于这里，因为它们处理样品中的气氛或真空。因此各 种新型的表面改性技术已被提出，它是基于，例如，在光反应性化合物12,13或激光烧蚀5,14，仅举几例。所提出的方法已经由Robertus 等人已很好地审查 。15，以及最近由Choi 等人的 16和中西17。
在这里，在这篇文章中，我们描述了一种新的原位表面修饰，这需要对二氧化钛的优势有机分子的光催化分解（ 二氧化钛）表面18,19协议。在该方法中，TiO 2膜被插入在玻璃基板和接口单元中的有机薄膜之间，且有机薄膜通过局部地照射紫外线（UV）的分解原位光到感兴趣的区域（λ<388纳米）。我们表明，新的协议可以用于创建细胞外基质蛋白和活细胞双方易地和原位微图案。 二氧化钛是生物相容的，化学稳定的，且光学透明的，特征，其中使得它友好引入在细胞培养实验。该协议提供了一个材料科学为基础的替代修改细胞培养支架的细胞培养环境。
Protocol
Representative Results
Discussion
在我们目前的协议， 二氧化钛膜通过射频磁控溅射形成。我们是有利于沉积的这种方法，因为它允许我们可重复使用亚纳米粗糙度调制成光催化二氧化钛薄膜。虽然溅射淀积过程是熟悉的材料科学家和电子工程师，它可能不是很访问生物学家。在这种情况下，旋涂的TiO 2膜 。将另外的选择23。在该方法中， 二氧化钛纳米颗粒溶解在溶剂中通过离心力铺展?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Glass coverslip | Warner Instruments | CS-15R15 | 15 mm, #1.5 thickness |
| Diamond scriber | Ogura Jewel Industry | D-Point Pen | |
| RF sputtering system | ANELVA | SPC350 | |
| TiO2 sputtering target | Kojundo Chemical Lab | Titanium (IV) oxide, target | Purity, 99.9% |
| Plasma reactor | Yamato | PR301 | |
| n-octadecyltrichlorosilane (OTS) |
Aldrich | 104817 | |
| Toluene | Wako | 204-01866 | |
| Tissue-culture dish (35 mm) | Greiner | 627160 | |
| Tissue-culture dish (60 mm) | BD Falcon | 353002 | |
| Type-IV collagen | Nitta Gelatin | Cellmatrix Type IV | |
| D-PBS | Gibco | 14190-144 | |
| Dulbecco's modified Eagle's medium (DMEM) | Gibco | 11885-084 | |
| Fetal bovine serum | Gibco | 12483-020 | Heat-inactivate and pass through a 0.22 mm filter before use |
| Horse serum | Gibco | 26050-088 | Pass through a 0.22 mm filter before use |
| Penicillin-streptomycin (100x) | Nacalai tesque | 26253-84 | |
| 7S nerve growth factor (NGF) | Alomone Labs | N-130 | |
| Bovine serum albumin (BSA) | Sigma | A2153 | |
| EDTA | Dojindo | N001 | Stock solution in 0.5 M |
| TiO2 nanoparticle | Tayca | TKD-701 |
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