Synthesis of photodegradable hydrogels as dynamically tunable cell culture platforms.
Synthesis of photodegradable hydrogels as dynamically tunable cell culture platforms.
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DOI：
10.1038/nprot.2010.139
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发表时间：
2010-12
期刊：
影响因子：
14.8
通讯作者：
中科院分区：
文献类型：
--
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我们描述了一个详细的程序，以创建基于光（乙二醇）的光（PEG）水凝胶并原位操纵材料特性。细胞相容的化学和降解过程可为2D或3D细胞培养的应用提供动态，可调节的变化。这些材料是通过合成基于O-亚硝基苯基的光降解单体而创建的，该单体可以耦合到原代胺。在这里，我们提供耦合程序以形成可光降解的交联链或纤连蛋白衍生的肽RGD形成光降解的固定链，以形成可光亮的系绳。在存在或不存在细胞的情况下，将水凝胶与可光降解的交联剂合成，从而可以直接封装或在表面上播种。可以通过时空控制凝胶微环境在2D或3D中探测细胞材料的相互作用，该环境可以进行独特的实验，以监视细胞对其利基变化的响应。通过低强度洪水照射（365至420 nm）的细胞相容性波长在几分钟内或高强度激光照射（405 nm）中，很容易实现降解。在此方案中，可光降解单体的合成和纯化大约需要2周，但可以通过购买O-硝基苯甲醚前体来大大缩短。用于水凝胶制造的无菌溶液的制备需要数小时，而形成最终水凝胶的反应在几分钟内完成。水凝胶降解发生在几秒钟到几分钟内，并以用户指导的光曝光发生。该综合方案对于在弹性基质上或内部的细胞培养过程中控制肽表现和底物模量很有用。这些基于PEG的材料可用于探测细胞细胞和细胞材料相互作用对2D或3D细胞功能的动态影响。虽然其他方案可用于控制肽表现或模量，但很少有人允许在原位操纵材料特性，并且在将细胞的存在下降低到千分尺。
We describe a detailed procedure to create photolabile, poly(ethylene glycol)-based (PEG) hydrogels and manipulate material properties in situ. The cytocompatible chemistry and degradation process enable dynamic, tunable changes for applications in 2D or 3D cell culture. The materials are created by synthesizing an o-nitrobenzylether-based photodegradable monomer that can be coupled to primary amines. Here, we provide coupling procedures to PEG-bis-amine to form a photodegradable crosslinker or to the fibronectin-derived peptide RGDS to form a photoreleasable tether. Hydrogels are synthesized with the photodegradable crosslinker in the presence or absence of cells, allowing direct encapsulation or seeding on surfaces. Cell-material interactions can be probed in 2D or 3D by spatiotemporally controlling the gel microenvironment, which allows unique experiments to be performed to monitor cell response to changes in their niche. Degradation is readily achieved with cytocompatible wavelengths of low intensity flood irradiation (365 to 420 nm) in minutes or with highintensity laser irradiation (405 nm) in seconds. In this protocol, synthesis and purification of the photodegradable monomers take approximately 2 weeks, but can be substantially shortened by purchasing the o-nitrobenzylether precursor. Preparation of the sterile solutions for hydrogel fabrication takes hours, while the reaction to form the final hydrogel is complete in minutes. Hydrogel degradation occurs on-demand, in seconds to minutes, with user-directed light exposure. This comprehensive protocol is useful for controlling peptide presentation and substrate modulus during cell culture on or within an elastic matrix. These PEG-based materials are useful for probing the dynamic influence of cell-cell and cell-material interactions on cell function in 2D or 3D. While other protocols are available for controlling peptide presentation or modulus, few allow manipulation of material properties in situ and in the presence of cells down to the micrometer scale.
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