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Probing the role of multicellular organization in three-dimensional microenvironments

Nature Methods volume 3, pages 369–375 (2006)Cite this article

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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Figure 1: Fabrication method and examples of DCP hydrogels.
Figure 2: DCP versatility in cell type, micropattern and hydrogel chemistry.
Figure 3: Quantitative control of cell microorganization within DCP hydrogels.
Figure 4: Multiorganization DCP hydrogels containing viable cells.
Figure 5: Regulation of matrix biosynthesis by chondrocyte cluster size.

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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.

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Authors and Affiliations

  1. Department of Bioengineering, University of California–San Diego, La Jolla, 92037, California, 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, 02139, Massachusetts, USA

    Dirk R Albrecht, Gregory H Underhill & Sangeeta N Bhatia

  3. Brigham and Women's Hospital, Boston, 02115, Massachusetts, USA

    Sangeeta N Bhatia

Authors
  1. Dirk R Albrecht
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  2. Gregory H Underhill
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Correspondence to Sangeeta N Bhatia.

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Supplementary information

Supplementary Fig. 1

DEP micropatterning of cells and microspheres (PDF 1450 kb)

Supplementary Fig. 2

Cell-cell forces in electric fields (PDF 174 kb)

Supplementary Fig. 3

Frequency dependence of relative DEP force and induced transmembrane voltage during DEP electropatterning (PDF 263 kb)

Supplementary Fig. 4

Fate of hydrogel-embedded chondrocytes over 14 days (PDF 845 kb)

Supplementary Fig. 5

Minimum separation distance between adjacent cells in patterned hydrogels (PDF 93 kb)

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. (MOV 749 kb)

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. (MOV 1326 kb)

Supplementary Methods (PDF 15 kb)

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Albrecht, D., Underhill, G., Wassermann, T. et al. Probing the role of multicellular organization in three-dimensional microenvironments. Nat Methods 3, 369–375 (2006). https://doi.org/10.1038/nmeth873

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