Bio-orthogonal tuning of matrix properties during 3D cell culture to induce morphological and phenotypic changes.

IF 13.1 1区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Hanyuan Gao, Mugdha Pol, Colette A Makara, Jiyeon Song, He Zhang, Xiaoyu Zou, Jamie M Benson, David L Burris, Joseph M Fox, Xinqiao Jia
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Abstract

Described herein is a protocol for producing a synthetic extracellular matrix that can be modified in situ during three-dimensional cell culture. The hydrogel platform is established using modular building blocks employing bio-orthogonal tetrazine (Tz) ligation with slow (norbornene, Nb) and fast (trans-cyclooctene, TCO) dienophiles. A cell-laden gel construct is created via the slow, off-stoichiometric Tz/Nb reaction. After a few days of culture, matrix properties can be altered by supplementing the cell culture media with TCO-tagged molecules through the rapid reaction with the remaining Tz groups in the network at the gel-liquid interface. As the Tz/TCO reaction is faster than molecular diffusion, matrix properties can be modified in a spatiotemporal fashion simply by altering the identity of the diffusive species and the diffusion time/path. Our strategy does not interfere with native biochemical processes nor does it require external triggers or a second, independent chemistry. The biomimetic three-dimensional cultures can be analyzed by standard molecular and cellular techniques and visualized by confocal microscopy. We have previously used this method to demonstrate how in situ modulation of matrix properties induces epithelial-to-mesenchymal transition, elicits fibroblast transition from mesenchymal stem cells and regulates myofibroblast differentiation. Following the detailed procedures, individuals with a bachelor's in science and engineering fields can successfully complete the protocol in 4-5 weeks. This protocol can be applied to model tissue morphogenesis and disease progression and it can also be used to establish engineered constructs with tissue-like anisotropy and tissue-specific functions.

在三维细胞培养过程中对基质特性进行生物正交调谐,以诱导形态和表型变化。
本文描述的是一种生产合成细胞外基质的方案,该基质可在三维细胞培养过程中进行原位修饰。水凝胶平台采用模块化构件,利用生物正交四嗪(Tz)与慢速二烯烃(降冰片烯,Nb)和快速二烯烃(反式环辛烯,TCO)连接。通过慢速、非均匀度的 Tz/Nb 反应,形成了细胞负载凝胶结构。培养几天后,通过在凝胶-液体界面上与网络中剩余的 Tz 基团快速反应,用 TCO 标记分子补充细胞培养基,可改变基质特性。由于 Tz/TCO 反应比分子扩散快,因此只需改变扩散物种的特性和扩散时间/路径,就能以时空方式改变基质特性。我们的策略不会干扰本地生化过程,也不需要外部触发器或第二种独立的化学方法。仿生三维培养物可以通过标准的分子和细胞技术进行分析,并通过共聚焦显微镜进行观察。我们以前曾用这种方法展示了原位调节基质特性如何诱导上皮向间充质转化、诱导间充质干细胞向成纤维细胞转化以及调节肌成纤维细胞分化。按照详细的程序,拥有理工科学士学位的人可以在 4-5 周内成功完成该方案。该方案可用于模拟组织形态发生和疾病进展,也可用于建立具有组织各向异性和组织特异功能的工程构建体。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Nature Protocols
Nature Protocols 生物-生化研究方法
CiteScore
29.10
自引率
0.70%
发文量
128
审稿时长
4 months
期刊介绍: Nature Protocols focuses on publishing protocols used to address significant biological and biomedical science research questions, including methods grounded in physics and chemistry with practical applications to biological problems. The journal caters to a primary audience of research scientists and, as such, exclusively publishes protocols with research applications. Protocols primarily aimed at influencing patient management and treatment decisions are not featured. The specific techniques covered encompass a wide range, including but not limited to: Biochemistry, Cell biology, Cell culture, Chemical modification, Computational biology, Developmental biology, Epigenomics, Genetic analysis, Genetic modification, Genomics, Imaging, Immunology, Isolation, purification, and separation, Lipidomics, Metabolomics, Microbiology, Model organisms, Nanotechnology, Neuroscience, Nucleic-acid-based molecular biology, Pharmacology, Plant biology, Protein analysis, Proteomics, Spectroscopy, Structural biology, Synthetic chemistry, Tissue culture, Toxicology, and Virology.
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