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Probing the role of multicellular organization in three-dimensional microenvironments

Nature Methods volume 3, pages 369375 (2006) | Download Citation

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Abstract

Successful application of living cells in regenerative medicine requires an understanding of how tissue structure relates to organ function. There is growing evidence that presentation of extracellular cues in a three-dimensional (3D) context can fundamentally alter cellular responses. Thus, microenvironment studies that previously were limited to adherent two-dimensional (2D) cultures may not be appropriate for many cell types. Here we present a method for the rapid formation of reproducible, high-resolution 3D cellular structures within a photopolymerizable hydrogel using dielectrophoretic forces. We demonstrate the parallel formation of >20,000 cell clusters of precise size and shape within a thin 2-cm2 hydrogel and the maintenance of high cell viability and differentiated cell markers over 2 weeks. By modulating cell-cell interactions in 3D clusters, we present the first evidence that microscale tissue organization regulates bovine articular chondrocyte biosynthesis. This platform permits investigation of tissue architecture in other multicellular processes, from embryogenesis to regeneration to tumorigenesis.

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Acknowledgements

We acknowledge B. Schumacher for help adapting biochemical assays, S. Khetani, T. Klein, M. Blewis, and M. Voegtline for helpful discussion and for providing materials, K. Jadin for providing imaging code, and J. Elisseeff and K. Anseth for hydrogel assistance. We thank K. Hudson and M. Akiyama for providing hepatocytes, M. Weiss and H. Strick-Marchand for providing BMEL cells, and S. Mittal for fabricating the branching array. Funding was provided by The Whitaker Foundation (D.R.A. fellowship), the US National Science Foundation, the National Institutes of Health, the David and Lucille Packard Foundation and the National Aeronautics and Space Administration.

Author information

Affiliations

  1. Department of Bioengineering, University of California–San Diego, La Jolla, California 92037, USA.

    • Dirk R Albrecht
    • , Gregory H Underhill
    • , Travis B Wassermann
    • , Robert L Sah
    •  & Sangeeta N Bhatia
  2. Harvard–Massachusetts Institute of Technology Division of Health Sciences and Technology & Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

    • Dirk R Albrecht
    • , Gregory H Underhill
    •  & Sangeeta N Bhatia
  3. Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

    • Sangeeta N Bhatia

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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Sangeeta N Bhatia.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    DEP micropatterning of cells and microspheres

  2. 2.

    Supplementary Fig. 2

    Cell-cell forces in electric fields

  3. 3.

    Supplementary Fig. 3

    Frequency dependence of relative DEP force and induced transmembrane voltage during DEP electropatterning

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    Supplementary Fig. 4

    Fate of hydrogel-embedded chondrocytes over 14 days

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    Supplementary Fig. 5

    Minimum separation distance between adjacent cells in patterned hydrogels

  6. 6.

    Supplementary Methods

Videos

  1. 1.

    Supplementary Video 1

    DEP micropatterning of living cells. This movie shows the simultaneous organization of fibroblasts within a 15% (wt/vol) PEG-DA polymer solution (viscosity: 3.3 cP), from an initially random field to an array of cell clusters. An a.c. bias of 3.0 Vrms, 3.0 MHz is applied at the onset of particle motion and remains for the duration of the movie. Under these conditions, cells aggregate by +DEP to high electric field strength at the electrodes. Elapsed time is displayed in seconds, indicating 10x real time playback and 60 s total patterning time. Electrodes are 100 μm apart.

  2. 2.

    Supplementary Video 2

    Simultaneous separation and micropatterning of cells and microspheres. This movie shows the separation and simultaneous patterning of 9.7 μm diameter polystyrene (PS) microspheres (dark border) and NIH 3T3 fibroblasts from a heterogeneous suspension. An a.c. bias of 5.3 Vrms, 3.0 MHz is applied at the onset of particle motion and remains for the duration of the movie. Under these conditions, cells aggregate by +DEP to high electric field strength at the electrodes. In contrast, microspheres pattern by −DEP to areas of low field strength, because PS has lower permittivity and conductivity than the medium. Elapsed time is displayed in seconds, indicating 4x real time playback and 20 s total patterning time. Electrodes are 100 μm apart.

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DOI

https://doi.org/10.1038/nmeth873

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