Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA.
Translational Musculoskeletal Research Center, Corporal Michael J. Crescenz VA Medical Center, Philadelphia, PA, USA.
Nat Protoc. 2022 Mar;17(3):618-648. doi: 10.1038/s41596-021-00652-9. Epub 2022 Feb 9.
Re-creating features of the native extracellular matrix (ECM) with engineered biomaterials has become a valuable tool to probe the influence of ECM properties on cellular functions (e.g., differentiation) and toward the engineering of tissues. However, characterization of newly secreted (nascent) matrix and turnover, which are important in the context of cells interacting with these biomaterials, has been limited by a lack of tools. We developed a protocol to visualize and quantify the spatiotemporal evolution of newly synthesized and deposited matrix by cells that are either cultured atop (2D) or embedded within (3D) biomaterial systems (e.g., hydrogels, fibrous matrices). This technique relies on the incorporation of a noncanonical amino acid (azidohomoalanine) into proteins as they are synthesized. Deposited nascent ECM components are then visualized with fluorescent cyclooctynes via copper-free cycloaddition for spatiotemporal analysis or modified with cleavable biotin probes for identification. Here we describe the preparation of hyaluronic acid hydrogels through ultraviolet or visible light induced cross-linking for 2D and 3D cell culture, as well as the fluorescent labeling of nascent ECM deposited by cells during culture. We also provide protocols for secondary immunofluorescence of specific ECM components and ImageJ-based ECM quantification methods. Hyaluronic acid polymer synthesis takes 2 weeks to complete, and hydrogel formation for 2D or 3D cell culture is performed in 2-3 h. Lastly, we detail the identification of nascent proteins, including enrichment, preparation and analysis with mass spectrometry, which can be completed in 10 d.
利用工程生物材料重现天然细胞外基质 (ECM) 的特征已成为一种有价值的工具，可用于研究 ECM 特性对细胞功能（例如分化）的影响，并有助于组织工程。然而，在细胞与这些生物材料相互作用的背景下，新分泌（初生）基质的特征描述和周转一直受到缺乏工具的限制。我们开发了一种方案，用于可视化和量化细胞在生物材料系统（例如水凝胶、纤维基质）上培养（2D）或嵌入（3D）时新合成和沉积的基质的时空演变。该技术依赖于在蛋白质合成时将非典型氨基酸（叠氮高丙氨酸）掺入蛋白质中。然后，通过无铜环加成反应用荧光环辛炔可视化新沉积的初生 ECM 成分，用于时空分析，或用可切割的生物素探针修饰以进行鉴定。在这里，我们描述了通过紫外光或可见光诱导交联来制备透明质酸水凝胶，用于 2D 和 3D 细胞培养，以及细胞在培养过程中沉积的初生 ECM 的荧光标记。我们还提供了用于特定 ECM 成分的二次免疫荧光和基于 ImageJ 的 ECM 定量方法的方案。透明质酸聚合物的合成需要 2 周的时间完成，而用于 2D 或 3D 细胞培养的水凝胶形成需要 2-3 小时。最后，我们详细介绍了初生蛋白质的鉴定，包括富集、准备和质谱分析，这可以在 10 天内完成。
