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

Autor: Gao, Hanyuan, Pol, Mugdha, Makara, Colette A., Song, Jiyeon, Zhang, He, Zou, Xiaoyu, Benson, Jamie M., Burris, David L., Fox, Joseph M., Jia, Xinqiao
Zdroj: Nature Protocols; 20240101, Issue: Preprints p1-52, 52p
Abstrakt: 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.
Databáze: Supplemental Index
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