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ma=86400 A multifunctional sensor for cell traction force, matrix remodeling and biomechanical assays in self-assembled 3D tissues in vitro | Nature Protocols
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A multifunctional sensor for cell traction force, matrix remodeling and biomechanical assays in self-assembled 3D tissues in vitro

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Abstract

Cell–matrix interactions, mediated by cellular force and matrix remodeling, result in dynamic reciprocity that drives numerous biological processes and disease progression. Currently, there is no available method for directly quantifying cell traction force and matrix remodeling in three-dimensional matrices as a function of time. To address this long-standing need, we developed a high-resolution microfabricated device that enables longitudinal measurement of cell force, matrix stiffness and the application of mechanical stimulation (tension or compression) to cells. Here a specimen comprising of cells and matrix self-assembles and self-integrates with the sensor. With primary fibroblasts, cancer cells and neurons we have demonstrated the feasibility of the sensor by measuring single or multiple cell force with a resolution of 1 nN and changes in tissue stiffness due to matrix remodeling by the cells. The sensor can also potentially be translated into a high-throughput system for clinical assays such as patient-specific drug and phenotypic screening. We present the detailed protocol for manufacturing the sensors, preparing experimental setup, developing assays with different tissues and for imaging and analyzing the data. Apart from microfabrication of the molds in a cleanroom (one time operation), this protocol does not require any specialized skillset and can be completed within 4–5 h.

Key points

  • Protocol for an in vitro sensor to measure cell traction force, matrix remodeling and biomechanical assays in self-assembled 3D tissues, including detailed steps for manufacturing the sensors, preparing the experimental setup, developing assays with different tissues and for imaging and analyzing the data.

  • This approach enables quantification for cells in a 3D environment with extracellular matrices around them, which more closely resembles the in vivo setting than 2D approaches.

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Fig. 1: A schematic illustration of the concept, design and functional mechanics of the sensor.
Fig. 2: Transfer of a point force applied on the ECM to the sensor (force calibration).
Fig. 3: Fabrication of the sensors and preparation of experimental setup.
Fig. 4: Detailed illustrations for tissue construction with cells in all parts of the tissue.
Fig. 5: Detailed illustration for tissue construction with cells inside the grips and the central region is cell free.
Fig. 6: Detailed illustration for tissue construction with two types of cells in two grips and the central region cell free.
Fig. 7: Various examples of tissues on the sensor.
Fig. 8: Application of the sensor for measurement of cell force, tissue stiffness and cell stretching.

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Data availability

All source data needed to validate findings in the protocol is available in the primary research papers and the Supplementary Materials of this paper. Any additional data related to this protocol may be requested from the authors. Source data are provided with this paper.

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Acknowledgements

Research reported in this publication was partially supported by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Number T32EB019944 to B.E. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Funding was also provided by the National Science Foundation grants ECCS 1934991 and CMMI 2342257, Mayo clinic grant PO 66236006 and Seed Grant from the Cancer Center at Illinois. M.T.A.S. is a CZ Biohub Investigator. We also thank the Paul Selvin lab for their assistance with the primary neurons, I. Choi for her assistance with PrCAFs, and A. Aly and J. Symanski for assistance with data analysis.

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Authors and Affiliations

  1. Department of Mechanical Science and Engineering, University of Illinois at Urbana–Champaign, Urbana, IL, USA

    Bashar Emon, Md Saddam Hossain Joy, William C. Drennan & M. Taher A. Saif

  2. CZ Biohub Chicago, LLC, Chicago, IL, USA

    M. Taher A. Saif

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  1. Bashar Emon
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Contributions

B.E. and M.T.A.S. conceived and designed the experiments. B.E., M.S.H.J. and W.C.D. performed the experiments, imaging and analysis. B.E., M.S.H.J., and M.T.A.S. prepared the manuscript. All authors have read and approved the final manuscript.

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Correspondence to M. Taher A. Saif.

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Nature Protocols thanks Yun Chen, Huw Colin-York, Qiang Wei, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Emon, B. et al. Sci. Adv. 7, eabf2629 (2021): https://doi.org/10.1126/sciadv.abf2629

Joy, M. S. H. et al. Proc. Natl Acad. Sci. 120, e2311995120 (2023): https://doi.org/10.1073/pnas.2311995120

Emon, B. et al. Acta Biomater. 173, 93–108 (2024): https://doi.org/10.1016/j.actbio.2023.11.015

Supplementary information

Supplementary Information

Supplementary Tutorial.

Supplementary Video 1

Calibration of force transmission in collagen I.

Supplementary Video 2

Phase contrast timelapse of neurite growth on the sensor. Adapted with permission from ref. 26, PNAS.

Supplementary Video 3

Fluorescence timelapse video of F-actin in a neuronal network on the sensor. Adapted with permission from ref. 26, PNAS.

Supplementary Video 4

Phase contrast timelapse of PrCAFs on the sensor for force measurement.

Supplementary Data 1

CAD file for mask design.

Source data

Source Data Fig. 2

Calibration of force transmission from the matrix to the sensor.

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Emon, B., Joy, M.S.H., Drennan, W.C. et al. A multifunctional sensor for cell traction force, matrix remodeling and biomechanical assays in self-assembled 3D tissues in vitro. Nat Protoc (2025). https://doi.org/10.1038/s41596-024-01106-8

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