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High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays

Nature Methods volume 8, pages 581–586 (2011)Cite this article

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Abstract

Heterogeneity in cell populations poses a major obstacle to understanding complex biological processes. Here we present a microfluidic platform containing thousands of nanoliter-scale chambers suitable for live-cell imaging studies of clonal cultures of nonadherent cells with precise control of the conditions, capabilities for in situ immunostaining and recovery of viable cells. We show that this platform mimics conventional cultures in reproducing the responses of various types of primitive mouse hematopoietic cells with retention of their functional properties, as demonstrated by subsequent in vitro and in vivo (transplantation) assays of recovered cells. The automated medium exchange of this system made it possible to define when Steel factor stimulation is first required by adult hematopoietic stem cells in vitro as the point of exit from quiescence. This technology will offer many new avenues to interrogate otherwise inaccessible mechanisms governing mammalian cell growth and fate decisions.

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Figure 1: Iso-osmotic perfusion microfluidic cell culture array.
Figure 2: Robust clonal cell culture in the microfluidic array.
Figure 3: Clonal heterogeneity of ND13 cells.
Figure 4: Maintenance of functional HSCs in microfluidic culture.
Figure 5: Culture of primary HSCs under dynamic conditions in microfluidic arrays.

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  • 03 June 2011

    In the version of this article initially published online, Michelle Miller's name was incorrect. The errors have been corrected for the PDF and HTML versions of this article.

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Acknowledgements

We thank M. Gasparetto for assistance with cell sorting and F. Kuchenbauer (Terry Fox Laboratory) for providing the ND13 cell line. This work was supported by grants from the Canadian Institutes of Health Research (MOP-93571 and RMF-82499) and the Natural Sciences and Engineering Research Council of Canada (RGPIN 312140). Infrastructure support was provided by the Canada Foundation for Innovation, Genome British Columbia, Western Diversification Canada and the Terry Fox Foundation. V.L. was supported by Natural Sciences and Engineering Research Council of Canada (Canada Graduate Scholarship) and the Michael Smith Foundation for Health Research (Junior Graduate Studentship). C.L.H. was supported by Michael Smith Foundation for Health Research (Biomedical Scholar) and Canadian Insitutes of Health Research (MSH-95337).

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Authors and Affiliations

  1. Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada

    Véronique Lecault, William Bowden, Asefeh Jarandehei & James M Piret

  2. Centre for High-Throughput Biology, University of British Columbia, Vancouver, British Columbia, Canada

    Michael VanInsberghe, William Bowden, Francis Viel, Thomas McLaughlin, Didier Falconnet, Adam K White & Carl L Hansen

  3. Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, British Columbia, Canada

    Véronique Lecault, Asefeh Jarandehei, Fariborz Taghipour & James M Piret

  4. Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada

    Sanja Sekulovic, David J H F Knapp, Stefan Wohrer, Michelle Miller, David G Kent, Michael R Copley, Connie J Eaves & R Keith Humphries

  5. Department of Physics and Astronomy, University of British Columbia, Vancouver, British Columbia, Canada

    William Bowden, Thomas McLaughlin & Carl L Hansen

  6. Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada

    Connie J Eaves

  7. Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada

    R Keith Humphries

Authors
  1. Véronique Lecault
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Contributions

V.L., S.S., M.M., D.J.H.F.K., S.W., C.J.E., R.K.H., J.M.P. and C.L.H. designed the research. V.L. performed microscale experiments. V.L., S.S., D.J.H.F.K., S.W. and M.M. performed macroscale cultures. S.S. performed in vivo assays. V.L., T.M., W.B. and C.L.H. designed and fabricated microfluidic cell culture arrays. V.L., M.V. and W.B. developed automated image acquisition. M.V., W.B. and F.V. wrote image and data analysis scripts. V.L., S.S., M.M., M.V., D.J.H.F.K., S.W. and W.B. analyzed data. A.J., J.M.P. and F.T. did modeling studies. V.L., T.M., W.B., D.F., A.K.W. and C.L.H. contributed to technology development. M.M., S.S., D.J.H.F.K., S.W., D.G.K., M.R.C., C.J.E. and R.K.H. provided cells, reagents and analytical tools. V.L., S.S., M.V., A.J., W.B., C.J.E., R.K.H., J.M.P. and C.L.H. wrote the manuscript.

Corresponding author

Correspondence to Carl L Hansen.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Notes 1–4 (PDF 2041 kb)

Supplementary Software 1

Image analysis scripts. (ZIP 18 kb)

Supplementary Video 1

Medium exchange does not disturb the spatial position of the cells. Cells were imaged continuously (1 frame s−1) during 10 min with and without medium exchange. (MOV 9139 kb)

Supplementary Video 2

Recovery of individual colonies. Colonies of ND13 cells were recovered from the microfluidic cell culture array using a micropipette after 72 h in culture. (MOV 7526 kb)

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Lecault, V., VanInsberghe, M., Sekulovic, S. et al. High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays. Nat Methods 8, 581–586 (2011). https://doi.org/10.1038/nmeth.1614

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