Capturing Complex Protein Gradients on Biomimetic Hydrogels for Cell-Based Assays
Capturing Complex Protein Gradients on Biomimetic Hydrogels for Cell-Based Assays
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DOI：
10.1002/adfm.200900968
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发表时间：
2009-11-09
影响因子：
19
通讯作者：
Lutolf, Matthias P.
中科院分区：
文献类型：
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作者：
Cosson, Steffen;Kobel, Stefan A.;Lutolf, Matthias P.
报道了一种通用策略，可以快速固定几乎任何所需蛋白质的复杂梯度在软聚乙二醇（PEG）水凝胶表面，使人想起天然细胞外基质（ECM）。微流控芯片用于生成生物素化或fc标记的融合蛋白的稳态梯度，这些融合蛋白在不到5分钟的时间内被NeutrAvidin或ProteinA捕获并结合到表面，并显示在表面上。结合方案的选择性和正交性使多种蛋白质形成平行和正交的重叠梯度，这在传统的细胞培养底物上是不可能的。在形成图案后，水凝胶从微流控芯片中释放出来，用于细胞培养。这种新颖的平台通过使用延时显微镜进行单细胞迁移实验进行了验证。细胞迁移的方向，以及原代人成纤维细胞的迁移速率，取决于固定纤维连接蛋白片段的浓度。这项技术可以很容易地应用于其他蛋白质，以解决不同细胞类型的丰富生物学问题。
A versatile strategy to rapidly immobilize complex gradients of virtually any desired protein on soft poly(ethylene glycol) (PEG) hydrogel surfaces that are reminiscent of natural extracellular matrices (ECM) is reported. A microfluidic chip is used to generate steady-state gradients of biotinylated or Fc-tagged fusion proteins that are captured and bound to the surface in less than 5 min by NeutrAvidin or ProteinA, displayed on the surface. The selectivity and orthogonality of the binding schemes enables the formation of parallel and orthogonal overlapping gradients of multiple proteins, which is not possible on conventional cell culture substrates. After patterning, the hydrogels are released from the microfluidic chip and used for cell culture. This novel platform is validated by conducting single-cell migration experiments using time-lapse microscopy. The orientation of cell migration, as well as the migration rate of primary human fibroblasts, depends on the concentration of an immobilized fibronectin fragment. This technique can be readily applied to other proteins to address a wealth of biological questions with different cell types.