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Spatially controlled simultaneous patterning of multiple growth factors in three-dimensional hydrogels

Abstract

Three-dimensional (3D) protein-patterned scaffolds provide a more biomimetic environment for cell culture than traditional two-dimensional surfaces, but simultaneous 3D protein patterning has proved difficult. We developed a method to spatially control the immobilization of different growth factors in distinct volumes in 3D hydrogels, and to specifically guide differentiation of stem/progenitor cells therein. Stem-cell differentiation factors sonic hedgehog (SHH) and ciliary neurotrophic factor (CNTF) were simultaneously immobilized using orthogonal physical binding pairs, barnase–barstar and streptavidin–biotin, respectively. Barnase and streptavidin were sequentially immobilized using two-photon chemistry for subsequent concurrent complexation with fusion proteins barstar–SHH and biotin–CNTF, resulting in bioactive 3D patterned hydrogels. The technique should be broadly applicable to the patterning of a wide range of proteins.

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Figure 1: Method for the simultaneous immobilization of SHH and CNTF.
Figure 2: 3D immobilization of barstar–SHH–488 using barnase–barstar.
Figure 3: 3D immobilization of biotin–CNTF–633 using biotin–streptavidin.
Figure 4: Representative figures for the simultaneous 3D patterning of biotin–CNTF–633 and barstar–SHH–488.
Figure 5: SHH and CNTF signalling pathways are activated in RPCs that are cultured on immobilized SHH and CNTF, respectively.
Figure 6: NPCs migrate into a channel of SHH with RGD.

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Acknowledgements

We are grateful to the Natural Sciences and Engineering Research Council (NSERC) and the Canadian Institutes of Health Research (CIHR) for funding through the Collaborative Health Research Program (CHRP, M.S.S., C.M.M.) and to CIHR for MOP-6279 (K.L.M.). We are also grateful to Le Fonds Québécoise de la recherche sur la nature et les technologies and Vision Science Research Program for financial support (R.G.W.). We thank members of the Shoichet research laboratory for discussions, particularly M. Cooke, S. Owen and K. Ho. We would also like to thank D. Bona for advice with protein expression and purification, R.G.W. Hartley for supplying a barnase expression plasmid (no 8621 pMT1002) through Addgene and J. F. Gauchat for gifting the CNTF plasmid with the biotinylation sequence.

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Contributions

R.G.W.—concept and design, fabrication of all gels, carried out all patterning experiments, collection and assembly of all data, data analysis and interpretation, manuscript writing, final approval of manuscript; S.A.—carried out, collected and analysed all bioactivity experiments; Y.A.—carried out all 3D cell migration studies; K.L.M.—concept and design for barstar–barnase, manuscript writing; C.M.M.—concept and design, data analysis and interpretation, manuscript writing; M.S.S.—concept and design, data analysis and interpretation, manuscript writing, final approval of manuscript.

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Correspondence to Molly S. Shoichet.

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Wylie, R., Ahsan, S., Aizawa, Y. et al. Spatially controlled simultaneous patterning of multiple growth factors in three-dimensional hydrogels. Nature Mater 10, 799–806 (2011). https://doi.org/10.1038/nmat3101

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