Faculty of Chemical Technology and Biotechnology , Budapest University of Technology and Economics , Műegyetem rkp. 3 , Budapest 1111 , Hungary.
Department of Biological Physics , Eötvös Loránd University , Pázmány Péter stny. 1A , Budapest 1117 , Hungary.
Langmuir. 2019 Feb 12;35(6):2412-2421. doi: 10.1021/acs.langmuir.8b03249. Epub 2019 Feb 1.
Micropatterning of living single cells and cell clusters over millimeter-centimeter scale areas is of high demand in the development of cell-based biosensors. Micropatterning methodologies require both a suitable biomimetic support and a printing technology. In this work, we present the micropatterning of living mammalian cells on carboxymethyl dextran (CMD) hydrogel layers using the FluidFM BOT technology. In contrast to the ultrathin (few nanometers thick in the dry state) CMD films generally used in label-free biosensor applications, we developed CMD layers with thicknesses of several tens of nanometers in order to provide support for the controlled adhesion of living cells. The fabrication method and detailed characterization of the CMD layers are also described. The antifouling ability of the CMD surfaces is demonstrated by in situ optical waveguide lightmode spectroscopy measurements using serum modeling proteins with different electrostatic properties and molecular weights. Cell micropatterning on the CMD surface was obtained by printing cell adhesion mediating cRGDfK peptide molecules (cyclo(Arg-Gly-Asp-d-Phe-Lys)) directly from aqueous solution using microchanneled cantilevers with subsequent incubation of the printed surfaces in the living cell culture. Uniquely, we present cell patterns with different geometries (spot, line, and grid arrays) covering both micrometer and millimeter-centimeter scale areas. The adhered patterns were analyzed by phase contrast microscopy and the adhesion process on the patterns was real-time monitored by digital holographic microscopy, enabling to quantify the survival and migration of cells on the printed cRGDfK arrays.
在基于细胞的生物传感器的发展中，对活的单细胞和细胞簇在毫米到厘米级大面积上的微图案化有很高的需求。微图案化方法既需要合适的仿生支持，也需要打印技术。在这项工作中，我们使用 FluidFM BOT 技术展示了活的哺乳动物细胞在羧甲基葡聚糖（CMD）水凝胶层上的微图案化。与通常用于无标记生物传感器应用的超薄（干燥状态下几纳米厚）CMD 薄膜不同，我们开发了几十纳米厚的 CMD 层，以提供对活细胞受控附着的支持。还描述了 CMD 层的制造方法和详细特性。通过使用具有不同静电特性和分子量的血清模拟蛋白进行原位光波导光模谱测量，证明了 CMD 表面的抗污能力。通过使用带有微通道的悬臂直接从水溶液中打印细胞黏附介导的 cRGDfK 肽分子（环（Arg-Gly-Asp-d-Phe-Lys）），在 CMD 表面上获得细胞微图案化，随后在活细胞培养中孵育打印表面。独特的是，我们展示了具有不同几何形状（点、线和网格阵列）的细胞图案，覆盖了微米和毫米到厘米级的大面积。通过相差显微镜对附着的图案进行分析，并通过数字全息显微镜实时监测图案上的附着过程，从而可以量化细胞在打印的 cRGDfK 阵列上的存活和迁移。
