Photocontrollable Dynamic Micropatterning of Non-adherent Mammalian Cells Using a Photocleavable Poly(ethylene glycol) Lipid
Photocontrollable Dynamic Micropatterning of Non-adherent Mammalian Cells Using a Photocleavable Poly(ethylene glycol) Lipid
复制标题
DOI：
10.1002/anie.201106106
复制
发表时间：
2012-01-01
影响因子：
16.6
通讯作者：
Nagamune, Teruyuki
中科院分区：
文献类型：
--
作者：
Yamaguchi, Satoshi;Yamahira, Shinya;Nagamune, Teruyuki
细胞微图案化已经成为一项重要的应用技术，应用范围从组织工程[1]和细胞微阵列[2]到细胞生物学的基础研究。[3]除了光刻、软光刻和喷墨打印等传统的图案化方法外，具有动态衬底的图案化方法目前是许多研究的重点，在动态衬底图案化方法中，细胞粘附性可以通过外部刺激，如热、[4]电压、[5]和光，[6]在任何所需的位置和任何时间点改变。这些时空图案化方法可以很容易地构建多个细胞系的图案，并有助于分析动态细胞活动。特别是，与热和电压相比，光可以很容易地以高空间和时间分辨率照射到传输空间的任何地方，并且光诱导对生物分子或活细胞的精细控制已被广泛报道，甚至在单分子水平上也是如此。[8B]因此，具有光响应底物的细胞图案化潜在地为生物学家提供了一种实用的工具。然而，几乎所有已报道的细胞微图案化方法在靶细胞上都有一个主要的限制。在传统的方法中，细胞的粘附性被用来将它们附着在裸露的或有配体涂层的表面上。因此，现有的方法不能应用于非贴壁细胞或弱贴壁细胞，包括血细胞(特别是免疫细胞)、一些癌细胞和干细胞。这些细胞系在生物和医学领域是重要的研究对象，因此，将当前微图案化方法的适用范围扩展到非贴壁细胞是一个重要的挑战。本文报告了一种可应用于非贴壁细胞的光诱导原位细胞微图案化方法。最近，我们报道了一种使用由聚乙二醇基和油基组成的细胞膜结合剂对非贴壁细胞进行构图的方法。[9]这种化合物可以与任何类型的细胞结合，而不会产生细胞毒性，因为油基部分可以以非共价方式插入到无处不在的脂质双层膜中。[9A]在本研究中，我们设计并合成了一种用于光诱导细胞构图的可光裂解的聚乙二醇脂，然后证实了细胞在涂有可光解的聚乙二醇脂的底物上的固定化受光照射剂量的调节。此外，本方法允许制备任意且精细的非贴壁细胞图案。此外，本发明的光响应衬底上的细胞微图案可以通过在期望的时间点上的光照射来改变。首先，我们设计并合成了一种可光解的聚乙二醇脂。在我们的设计中，在聚乙二醇和油基之间加入了一个可光切割的单元，并在聚乙二醇链的另一端添加了一个氨基反应酯基团，以便通过酰胺偶联反应附着到底物上(图1a)。涂层后，油基部分预计会暴露并锚定活细胞(图1b)。此外，这种分子可以通过辐射裂解，然后聚乙二醇部分暴露在光照射的区域(图1b)。据报道，聚乙二醇涂层的表面会抑制细胞的黏附。[10]因此，细胞黏附和非黏附的表面有望通过光照射来制备(图1b)。一种可光解的聚乙二醇脂由一种商业上可获得的邻硝基苄基光解连接物[11]合成，并用标准方法进行了表征(见支持信息)。用~1H核磁共振波谱证实了…辐照后聚乙二醇脂的光解性质
Cell micropatterning has become an important technology for a wide variety of applications, ranging from tissue engineering [1] and cell microarrays [2] to fundamental studies in cell biology.[3] In addition to conventional patterning methods, such as photolithography, soft lithography, and inkjet printing, patterning methods with dynamic substrates, in which cell adhesive properties can be changed by an external stimulus, such as heat,[4] voltage,[5] and light,[6] at any desired position and any point in time, are currently the focus of many studies. These spatiotemporal patterning methods can easily construct patterns of multiple cell lines and be useful for analyzing dynamic cellular activity.[7] In particular, in contrast to heat and voltage, light can be readily applied to anywhere in transmissive spaces with high spatial and temporal resolution, and light-induced fine control of biomolecules or living cells has been widely reported,[6, 8] even at a single-molecular level.[8b] Therefore, cell patterning with light-responsive substrates potentially offers a practical tool for biologists. However, almost all reported cell micropatterning methods have a major limitation in target cells. In conventional methods, the adhesiveness of cells is used to attach them onto bare or ligand-coated surfaces. Therefore, the existing methods cannot be applied to non-or weakly adherent cells, which include blood cells (especially immunocytes), some cancer cells, and stem cells. These cell lines are important as research targets in biological and medical fields, and for this reason expansion of an applicable range of current micropatterning methods to non-adherent cells is an important challenge.We report herein a light-induced in situ cell micropatterning method that can be applied to non-adherent cells. Recently, we reported a cell patterning method for nonadherent cells using a cell membrane binding reagent consisting of poly (ethylene glycol)(PEG) and an oleyl group.[9] This compound can bind to any type of cell without cytotoxicity, because the oleyl moiety can be inserted into ubiquitous lipid bilayer membranes in a noncovalent manner.[9a] In the current study, a photocleavable PEG-lipid was newly designed and synthesized for light-induced cell patterning, and then cell immobilization on the substrate coated with the photocleavable PEG-lipid was confirmed to be regulated by the dose of light exposure. Moreover, the present method allows the preparation of arbitrary and fine patterns of non-adherent cells. Furthermore, the cell micropattern on the present light-responsive substrate can be altered by light irradiation at a desired point in time. First, we designed and synthesized a photocleavable PEG-lipid. In our design, a photocleavable unit was incorporated between the PEG and oleyl moieties, and at the opposite end of the PEG segment an amino-reactive ester group was added for attachment onto the substrate through an amide coupling reaction (Figure 1a). After coating, the oleyl moieties are expected to be exposed and to anchor living cells (Figure 1 b). Moreover, this molecule can be cleaved by irradiation, and then the PEG moiety is exposed at the light-irradiated area (Figure 1b). It has been reported that a PEG-coated surface inhibits cell adhesion.[10] Therefore, cell-adhesive and nonadhesive surfaces were expected to be prepared by light irradiation (Figure1b). A photocleavable PEG-lipid was synthesized from a commercially available o-nitrobenzyl photocleavable linker [11] and characterized by using standard methods (see the Supporting Information). The photolytic property of the PEG-lipid in solution was confirmed by means of 1H NMR spectroscopy after irradiation with …