Protocol | Published:

Cell-derived matrices for studying cell proliferation and directional migration in a complex 3D microenvironment

Nature Protocols volume 12, pages 23762390 (2017) | Download Citation

Abstract

2D surfaces offer simple analysis of cells in culture, yet these often yield different cell morphologies and responses from those observed in vivo. Considerable effort has therefore been expended on the generation of more tissue-like environments for the study of cell behavior in vitro. Purified matrix proteins provide a 3D scaffold that better mimics the in vivo situation; however, these are far removed from the complex tissue composition seen in vivo. Cell-derived matrices (CDMs) offer a more physiologically relevant alternative for studying in vivo-like cell behavior in vitro. In the protocol described here, fibroblasts cultured on gelatin-coated surfaces are maintained in the presence of ascorbic acid to strengthen matrix deposition over 1–3 weeks. The resulting fibrillar CDMs are denuded of cells, and other cells are subsequently cultured on them, after which their behavior is monitored. We also demonstrate how to use CDMs as an in vivo-relevant reductionist model for studying tumor-stroma-induced changes in carcinoma cell proliferation and migration.

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Acknowledgements

M. Georgiadou (University of Turku Centre for Biotechnology) is acknowledged for providing the bright-field images of CDMs presented in Figure 3. J. Norman (Beatson Institute for Cancer Research) is acknowledged for providing the TIFs. P. Caswell (University of Manchester) and J.F. Marshall (Barts Cancer Institute) are acknowledged for the A2780 and the MCF10 DCIS.COM cell lines, respectively. The Turku Centre for Biotechnology Imaging Core facility is acknowledged for help with the imaging. We gratefully acknowledge the following funding sources: Academy of Finland, European Research Council Consolidator Grant (no. 615258), the Sigrid Juselius Foundation and the Finnish Cancer Organization to J.I. R.K. was funded by the University of Turku/TUBS TuDMM doctoral program and G.J. was funded by an EMBO LTF.

Author information

Author notes

    • Riina Kaukonen
    •  & Guillaume Jacquemet

    These authors contributed equally to this work.

Affiliations

  1. Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland.

    • Riina Kaukonen
    • , Guillaume Jacquemet
    • , Hellyeh Hamidi
    •  & Johanna Ivaska
  2. Department of Biochemistry, University of Turku, Turku, Finland.

    • Johanna Ivaska

Authors

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  2. Search for Guillaume Jacquemet in:

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Contributions

J.I., G.J., H.H. and R.K. organized and wrote the manuscript. H.H. provided the illustrated workflow and edited the manuscript. G.J. and R.K. produced the data. R.K. produced the video-guided material with help from G.J and J.I.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Johanna Ivaska.

Supplementary information

Videos

  1. 1.

    Workflow

    This video outlines the entire workflow used to generate CDMs.

  2. 2.

    Coverslip preparation

    This video shows how to prepare gelatin-coated coverslips (Steps 1–6 of the protocol).

  3. 3.

    Plating of fibroblasts

    This video shows how to plate fibroblasts for CDM production (Steps 7–10 of the protocol).

  4. 4.

    Extraction of CDMs.

    This video shows how to extract CDMs (Steps 11–17 of the protocol).

  5. 5.

    Staining of CDMs.

    This video shows how to stain CDMs using immunofluorescence (Step 18A of the protocol).

  6. 6.

    Analysis of cell proliferation.

    This video shows how to analyze cell proliferation on CDMs using an IncuCyte Zoom live-cell incubator (Step 18B of the protocol).

  7. 7.

    Quantification of cell migration.

    This video shows how to quantify cell migration on CDMs using ImageJ and the Chemotaxis tool (Step 18C of the protocol).

  8. 8.

    Migration of an ovarian carcinoma cell.

    This video shows an ovarian carcinoma cell transiently expressing Lifeact RFP (to visualize the actin cytoskeleton) migrating on TIF CDMs (labeled in green using Alexa Fluor 488 recombinant fibronectin). Images were acquired on a spinning-disk microscope using a 63× objective.

About this article

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DOI

https://doi.org/10.1038/nprot.2017.107

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