Dorsey Taylor B, Grath Alexander, Xu Cancan, Hong Yi, Dai Guohao
Department of Biomedical Engineering, Rensselaer Polytechnic Institute; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute; Department of Bioengineering, Northeastern University.
Department of Biomedical Engineering, Rensselaer Polytechnic Institute; Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute.
J Vis Exp. 2017 Sep 15(127):55873. doi: 10.3791/55873.
There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.
有许多生物刺激可以影响细胞行为和干细胞分化。一般的细胞培养方法依赖于培养基中的可溶性因子来控制细胞行为。然而，添加可溶性物质无法模拟某些信号基序，如基质结合生长因子、细胞间信号传导和空间生化线索，而这些对细胞来说是常见的影响因素。此外，基质的生物物理特性，如底物硬度，在细胞命运中起着重要作用，这是传统细胞培养方法难以操控的。在本方法中，我们描述了一种使用光化学在合成聚乙二醇（PEG）水凝胶上提供图案化生物活性蛋白的简单方案。该平台允许独立控制底物硬度和空间生化线索。这些水凝胶可以实现大范围的生理相关硬度值。此外，这些水凝胶的表面可以通过硫醇-烯点击化学反应与生物活性肽或蛋白质进行光图案化。这些方法已经过优化，以在表面固定后保留蛋白质功能。这是一个通用方案，可应用于任何感兴趣的蛋白质或肽，以创建各种图案。最后，接种在这些生物活性水凝胶表面的细胞在对空间特异性信号作出反应时，可以随时间进行监测。
