Mohammed Danahe, Pardon Gaspard, Versaevel Marie, Bruyère Céline, Alaimo Laura, Luciano Marine, Vercruysse Eléonore, Pruitt Beth L, Gabriele Sylvain
Laboratory for Complex Fluids and Interfaces, Mechanobiology and Soft Matter Group, Research Institute for Biosciences, University of Mons, Place du Parc, 20, 7000 Mons, Belgium.
Departments of Bioengineering and Mechanical Engineering, Microsystems Laboratory, Stanford University, 452 Escondido Mail, Stanford, CA 94305-4040 USA.
Cell Mol Bioeng. 2019 Sep 25;13(1):87-98. doi: 10.1007/s12195-019-00600-4. eCollection 2020 Feb.
The orientation of collagen fibers in native tissues plays an important role in cell signaling and mediates the progression of tumor cells in breast cancer by a contact guidance mechanism. Understanding how migration of epithelial cells is directed by the alignment of collagen fibers requires assays with standardized orientations of collagen fibers.
To address this issue, we produced micro-stripes with aligned collagen fibers using an easy-to-use and versatile approach based on the aspiration of a collagen solution within a microchannel. Glass coverslips were functionalized with a (3-aminopropyl)triethoxysilane/glutaraldehyde linkage to covalently anchor micro-stripes of aligned collagen fibers, whereas microchannels were functionalized with a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) nonionic triblock polymer to prevent adhesion of the collagen micro-stripes.
Using this strategy, microchannels can be peeled off to expose micro-stripes of aligned collagen fibers without affecting their mechanical integrity. We used time-lapse confocal reflection microscopy to characterize the polymerization kinetics of collagen networks for different concentrations and the orientation of collagen fibers as a function of the microchannel width. Our results indicate a non-linear concentration dependence of the area of fluorescence, suggesting that the architecture of collagen networks is sensitive to small changes in concentration. We show the possibility to influence the collagen fibril coverage by adjusting the concentration of the collagen solution.
We applied this novel approach to study the migration of epithelial cells, demonstrating that collagen micro-stripes with aligned fibers represent a valuable assay for studying cell contact guidance mechanisms.
天然组织中胶原纤维的取向在细胞信号传导中起重要作用，并通过接触导向机制介导乳腺癌中肿瘤细胞的进展。了解上皮细胞的迁移如何由胶原纤维的排列引导需要使用具有标准化胶原纤维取向的检测方法。
为了解决这个问题，我们基于在微通道内抽吸胶原溶液的简单通用方法，制备了具有排列胶原纤维的微条纹。玻璃盖玻片用（3-氨丙基）三乙氧基硅烷/戊二醛连接进行功能化，以共价固定排列胶原纤维的微条纹，而微通道用聚（环氧乙烷）-聚（环氧丙烷）-聚（环氧乙烷）（PEO-PPO-PEO）非离子三嵌段聚合物进行功能化，以防止胶原微条纹的粘附。
使用这种策略，可以剥离微通道以暴露排列胶原纤维的微条纹，而不会影响其机械完整性。我们使用延时共聚焦反射显微镜来表征不同浓度下胶原网络的聚合动力学以及胶原纤维的取向作为微通道宽度的函数。我们的结果表明荧光面积的浓度依赖性是非线性的，这表明胶原网络的结构对浓度的微小变化敏感。我们展示了通过调节胶原溶液的浓度来影响胶原原纤维覆盖率的可能性。
我们应用这种新方法来研究上皮细胞的迁移，证明具有排列纤维的胶原微条纹是研究细胞接触导向机制的有价值的检测方法。
