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Arrayed cellular microenvironments for identifying culture and differentiation conditions for stem, primary and rare cell populations

Nature Protocols volume 7, pages 703–717 (2012)Cite this article

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Abstract

During the development of an organism, cells are exposed to a myriad of signals, structural components and scaffolds, which collectively make up the cellular microenvironment. The majority of current developmental biology studies examine the effect of individual or small subsets of molecules and parameters on cellular behavior, and they consequently fail to explore the complexity of factors to which cells are exposed. Here we describe a technology, referred to as arrayed cellular microenvironments (ACMEs), that allows for a high-throughput examination of the effects of multiple extracellular components in a combinatorial manner on any cell type of interest. We will specifically focus on the application of this technology to human pluripotent stem cells (hPSCs), a population of cells with tremendous therapeutic potential, and one for which growth and differentiation conditions are poorly characterized and far from defined and optimized. A standard ACME screen uses the technologies previously applied to the manufacture and analysis of DNA microarrays, requires standard cell-culture facilities and can be performed from beginning to end within 5–10 days.

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Figure 1: Flowchart outlining the protocol.
Figure 2
Figure 3: Example of a live-analysis experiment to identify conditions that promote hPSC attachment and growth.
Figure 4: Characterization of printed ACME slides.
Figure 5: Example of an endpoint analysis experiment.

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Acknowledgements

D.A.B. was supported by funding from the University of California San Diego Stem Cell Program and by a gift from Michael and Nancy Kaehr. This research was supported in part by the California Institute of Regenerative Medicine (RS1-00172-1) to S.C. and (RB1-01406) to K.W.

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Authors and Affiliations

  1. Cellular and Molecular Medicine, Stem Cell Program, University of California, San Diego, California, USA

    David A Brafman & Karl Willert

  2. Department of Bioengineering, University of California, San Diego, California, USA

    Shu Chien

Authors
  1. David A Brafman
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  2. Shu Chien
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  3. Karl Willert
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Contributions

D.A.B., S.C. and K.W. developed the protocol. D.A.B. and K.W. designed and performed the experiments. D.A.B., S.C. and K.W. analyzed the results. D.A.B., S.C. and K.W. wrote the manuscript.

Corresponding authors

Correspondence to David A Brafman or Karl Willert.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig 1

Schematic representation of ACME format. (a) Schematic of a 10x10 subarray that contains 20 different conditions spotted in replicates of five. Spot diameter (closed circles) is 150 μm and the center to center distance between neighboring spots is 450 μm. These subarrays can be arranged to generate several different designs including (b) a 8x2, 1600 spot/320 condition, (c) a 16x4, 6400 spot/1280 condition, or (d) a 16x5, 8000 spot/1600 condition array. (PPT 415 kb)

Supplementary Table 1

Example of analysis of raw data generated from the array in Figure 5e. (DOC 1214 kb)

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Brafman, D., Chien, S. & Willert, K. Arrayed cellular microenvironments for identifying culture and differentiation conditions for stem, primary and rare cell populations. Nat Protoc 7, 703–717 (2012). https://doi.org/10.1038/nprot.2012.017

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