细胞外基质
组织工程
机械生物学
基质(化学分析)
化学
计算生物学
代谢工程
细胞生物学
生物信息学
生物化学
计算机科学
细胞
生物
酶
色谱法
遗传学
作者
Claudia Loebel,Aya M. Saleh,Kathryn R. Jacobson,Ryan Daniels,Robert L. Mauck,Sarah Calve,Jason A. Burdick
出处
期刊:Nature Protocols
[Springer Nature]
日期:2022-02-09
卷期号:17 (3): 618-648
被引量:16
标识
DOI:10.1038/s41596-021-00652-9
摘要
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. When cells are grown in or on a biomaterial, they continue to produce a natural extracellular matrix (ECM). This protocol describes the analysis of nascent ECM proteins using metabolic labeling, click chemistry, confocal microscopy and mass spectrometry.
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