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Immobilization of growth factors on solid supports for the modulation of stem cell fate

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Abstract

Surface- and matrix-bound signals modulate stem cell fate in vivo and in vitro. This protocol enables the immobilization of a wide range of biomolecules that contain primary amino groups to different types of solid carriers, including glass substrates and standard polystyrene well plates. We describe how thin polymer coatings of poly(octadecene-alt-maleic anhydride) can be used to covalently attach growth factors directly, or through poly(ethylene glycol) spacers, to solid supports at defined concentrations. Surface-immobilized growth factors can be presented over a wide range of concentrations (5–150 ng cm−2), as we have previously shown for leukemia inhibitory factor and stem cell factor. Cell activation can be achieved in the presence of adhesion-promoting extracellular matrix proteins. Depending on the methods used, the overall procedure takes 1.5–3 d. In general, the approach can be used to investigate the effect of defined amounts of immobilized growth factors on stem cells and on the maintenance, growth and differentiation of other cell types.

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Figure 1: Two immobilization schemes for the covalent immobilization of functional growth factors by means of copolymer coatings.
Figure 2
Figure 3: Reactive conversions involved in the tethering of biomolecules to poly(octadecene-alt-maleic anhydride) films: (a) initial state resulting from the covalent attachment of polymer film to amine-functionalized glass substrate, (b) immobilization of growth factors (e.g., LIF) through reaction of the primary amino groups of lysine side chains to the anhydride groups of the polymer, (c) immobilization of growth factors to carboxylic acid groups of the polymer-bound PEG spacer.
Figure 4: The custom-built immobilization chamber used in the protocol to allow defined immobilization of growth factors on POMA-coated glass slides ((a) disassembled, (b) assembled for protein immobilization).
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Acknowledgements

We thank T. Lenk for technical assistance. The financial support of the Federal Ministry of Science and Education of Germany and the German Research Foundation is gratefully acknowledged.

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Author notes

  1. Tilo Pompe and Katrin Salchert: These authors contributed equally to this work.

Authors and Affiliations

  1. Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials, Dresden, Germany

    Tilo Pompe, Kristin Alberti & Carsten Werner

  2. Lausitz University of Applied Sciences, Senftenberg, Germany

    Katrin Salchert

  3. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada

    Peter Zandstra

  4. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Canada

    Peter Zandstra

  5. Technische Universität Dresden, Center for Regenerative Therapies, Dresden, Germany

    Carsten Werner

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  1. Tilo Pompe
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  2. Katrin Salchert
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  3. Kristin Alberti
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  4. Peter Zandstra
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  5. Carsten Werner
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Contributions

T.P. and K.S. designed and conducted the experiments, analyzed the data and wrote the paper; K.A. designed and conducted the experiments and analyzed the data; P.Z. and C.W. designed the experiments and wrote the paper.

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Correspondence to Carsten Werner.

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The authors declare no competing financial interests.

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Pompe, T., Salchert, K., Alberti, K. et al. Immobilization of growth factors on solid supports for the modulation of stem cell fate. Nat Protoc 5, 1042–1050 (2010). https://doi.org/10.1038/nprot.2010.70

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