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Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels

Nature Protocols volume 7, pages 1247–1259 (2012)Cite this article

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Abstract

This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel-incorporating chambers between surface-accessible microchannels. By using this platform, well-defined biochemical and biophysical stimuli can be applied to multiple cell types interacting over distances of <1 mm, thereby replicating many aspects of the in vivo microenvironment. Capabilities exist for time-dependent manipulation of flow and concentration gradients as well as high-resolution real-time imaging for observing spatial-temporal single-cell behavior, cell-cell communication, cell-matrix interactions and cell population dynamics. These heterotypic cell type assays can be used to study cell survival, proliferation, migration, morphogenesis and differentiation under controlled conditions. Applications include the study of previously unexplored cellular interactions, and they have already provided new insights into how biochemical and biophysical factors regulate interactions between populations of different cell types. It takes 3 d to fabricate the system and experiments can run for up to several weeks.

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Figure 1: Schematic and photograph of the microfluidic cell culture assay.
Figure 2: Photolithography and soft lithography procedures.
Figure 3: Procedure for hydrogel-incorporating microfluidic assay preparation.
Figure 4: Using collagen.
Figure 5: Cell culture in microfluidic assay.
Figure 6: Characterization of the diffusion profile of applied VEGF.
Figure 7: Confocal images of 3D angiogenic response into the type 1 collagen (2.0 mg ml−1, polymerized at pH 7.4) induced by VEGF diffused from the right channel.
Figure 8: 3D interaction of various types of cells with incorporated hydrogel.
Figure 9
Figure 10: Examples of cell-cell 3D interaction in hydrogel.

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Acknowledgements

We acknowledge support to S.C. from the National Research Foundation of Korea (grant no. 2010-0023975), to R.S. from Japan Science and Technology Agency and Japan Society for Promotion of Science (22680037, G2212) and to R.D.K. from the National Science Foundation (CBET-0939511). We thank Y. Kikkawa, Tokyo University of Pharmacy and Life Sciences, for generously providing anti-CD146 antibody.

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Authors and Affiliations

  1. School of Mechanical Engineering, Korea University, Seoul, Korea

    Yoojin Shin, Sewoon Han & Seok Chung

  2. Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, M

    Jessie S Jeon, Ioannis K Zervantonakis & Roger D Kamm

  3. assachusetts, USA

    Jessie S Jeon, Ioannis K Zervantonakis & Roger D Kamm

  4. Department of System Design Engineering, Keio University, Yokohama, Japan

    Kyoko Yamamoto & Ryo Sudo

  5. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, M

    Roger D Kamm

  6. assachusetts, USA

    Roger D Kamm

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  1. Yoojin Shin
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Contributions

Y.S. and S.H. equally contributed to this protocol, designing and carrying out the experiments and writing the paper. J.S.J., K.Y., I.K.Z., R.S. and S.C. designed and carried out the experiments. S.C. is responsible for all the experiments described in this article and preparation of the paper. R.D.K. is responsible for providing guidance for the experiments and for editing the paper.

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Correspondence to Roger D Kamm or Seok Chung.

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Shin, Y., Han, S., Jeon, J. et al. Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels. Nat Protoc 7, 1247–1259 (2012). https://doi.org/10.1038/nprot.2012.051

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