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<p>Elucidating the mechanical regulation of angiogenesis remains a challenge owing to the complexities of measuring cellular forces in this dynamic, multicellular and three-dimensional (3D) process. Current methods for force measurements typically involve traction force microscopy (TFM) applied to single cells or monolayers on 2D substrates or to individual cells within 3D extracellular matrix (ECM)-like gels. Here we present a protocol for mimicking and imaging dynamic early angiogenic sprouting into biomimetic matrices compatible with 3D TFM and for visualizing matrix degradation. Given that reliably acquiring sufficiently large 3D TFM datasets in multicellular systems is challenging, our protocol emphasizes best practices for higher-throughput data acquisition and for accurately imaging, analyzing and interpreting cell–ECM forces using our open-source TFMLAB software. As such, this assay provides a defined and reproducible system to study cellular forces and matrix degradation during angiogenesis in response to perturbations of cell-intrinsic signaling and mixed cell populations, as well as ECM cues. We further provide protocols for immunofluorescence analysis of angiogenic sprouts formed within the matrices and their retrieval from the hydrogel for downstream sequencing. Depending on the number of samples, sample preparation can take between 2 h and 4 h followed by a 15–17 h overnight wait time for angiogenic invasion. The 3D TFM data acquisition can take 2–6 h, while downstream processing of samples can take either 1 h (endothelial cell isolation) or up to 5 d (immunofluorescence). Notably, this workflow demands minimal prior expertise in programming, biophysics or molecular biology.</p>

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Investigation of mechanical forces during multicellular early angiogenic sprouting by three-dimensional traction force microscopy in hydrogel matrices

  • 期刊论文

  • 发表日期：

    2026年1月23日

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  • Apeksha Shapeti,
  • Janne de Jong,
  • Jorge Barrasa-Fano,
  • José Antonio Sanz-Herrera,
  • Eva Faurobert,
  • Hans Van Oosterwyck

摘要

Elucidating the mechanical regulation of angiogenesis remains a challenge owing to the complexities of measuring cellular forces in this dynamic, multicellular and three-dimensional (3D) process. Current methods for force measurements typically involve traction force microscopy (TFM) applied to single cells or monolayers on 2D substrates or to individual cells within 3D extracellular matrix (ECM)-like gels. Here we present a protocol for mimicking and imaging dynamic early angiogenic sprouting into biomimetic matrices compatible with 3D TFM and for visualizing matrix degradation. Given that reliably acquiring sufficiently large 3D TFM datasets in multicellular systems is challenging, our protocol emphasizes best practices for higher-throughput data acquisition and for accurately imaging, analyzing and interpreting cell–ECM forces using our open-source TFMLAB software. As such, this assay provides a defined and reproducible system to study cellular forces and matrix degradation during angiogenesis in response to perturbations of cell-intrinsic signaling and mixed cell populations, as well as ECM cues. We further provide protocols for immunofluorescence analysis of angiogenic sprouts formed within the matrices and their retrieval from the hydrogel for downstream sequencing. Depending on the number of samples, sample preparation can take between 2 h and 4 h followed by a 15–17 h overnight wait time for angiogenic invasion. The 3D TFM data acquisition can take 2–6 h, while downstream processing of samples can take either 1 h (endothelial cell isolation) or up to 5 d (immunofluorescence). Notably, this workflow demands minimal prior expertise in programming, biophysics or molecular biology.