Kandel Judith, Lee Hyun-Su, Sobolewski Peter, Tomczyk Nancy, Composto Russell J, Eckmann David M
Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
Department of Materials Science and Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
Biosens Bioelectron. 2014 Aug 15;58:249-257. doi: 10.1016/j.bios.2014.02.053. Epub 2014 Feb 28.
Traditionally, fibronectin has been used as a physisorbed surface coating (physFN) in cell culture experiments due to its critical role in cell adhesion. However, because the resulting layer is thick, unstable, and of unpredictable uniformity, this method of fibronectin deposition is unsuitable for some types of research, including quartz crystal microbalance (QCM) experiments involving cells. Here, we present a new method for chemical immobilization of fibronectin onto silicon oxide surfaces, including QCM crystals pre-coated with silicon oxide. We characterize these chemically coated fibronectin surfaces (chemFN) as well as physFN ones using spectroscopic ellipsometry (SE), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and contact angle measurements. A cell culture model demonstrates that cells on chemFN and physFN surfaces exhibit similar viability, structure, adhesion and metabolism. Finally, we perform QCM experiments using cells on both surfaces which demonstrate the superior suitability of chemFN coatings for QCM research, and provide real-time QCM-D data from cells subjected to an actin depolymerizing agent. Overall, our method of chemical immobilization of fibronectin yields great potential for furthering cellular experiments in which thin, stable and uniform coatings are desirable. As QCM research with cells has been rather limited in success thus far, we anticipate that this new technique will particularly benefit this experimental system by availing it to the much broader field of cell mechanics.
传统上，纤连蛋白因其在细胞黏附中的关键作用，在细胞培养实验中一直被用作物理吸附的表面涂层（物理吸附纤连蛋白）。然而，由于形成的涂层较厚、不稳定且均匀性不可预测，这种纤连蛋白沉积方法不适用于某些类型的研究，包括涉及细胞的石英晶体微天平（QCM）实验。在此，我们提出一种将纤连蛋白化学固定在氧化硅表面的新方法，包括预先涂覆氧化硅的QCM晶体。我们使用光谱椭偏仪（SE）、傅里叶变换红外光谱（FTIR）、原子力显微镜（AFM）和接触角测量对这些化学涂层的纤连蛋白表面（化学固定纤连蛋白）以及物理吸附纤连蛋白表面进行了表征。细胞培养模型表明，化学固定纤连蛋白表面和物理吸附纤连蛋白表面上的细胞具有相似的活力、结构、黏附力和代谢。最后，我们对两种表面上的细胞进行了QCM实验，结果表明化学固定纤连蛋白涂层在QCM研究中具有更好的适用性，并提供了来自受到肌动蛋白解聚剂作用的细胞的实时QCM-D数据。总体而言，我们的纤连蛋白化学固定方法在推进需要薄、稳定且均匀涂层的细胞实验方面具有巨大潜力。由于迄今为止细胞QCM研究的成功案例相当有限，我们预计这项新技术将特别有益于这个实验系统，使其能够应用于更广泛的细胞力学领域。
