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In situ cell manipulation through enzymatic hydrogel photopatterning

Abstract

The physicochemical properties of hydrogels can be manipulated in both space and time through the controlled application of a light beam. However, methods for hydrogel photopatterning either fail to maintain the bioactivity of fragile proteins and are thus limited to short peptides, or have been used in hydrogels that often do not support three-dimensional (3D) cell growth. Here, we show that the 3D invasion of primary human mesenchymal stem cells can be spatiotemporally controlled by micropatterning the hydrogel with desired extracellular matrix (ECM) proteins and growth factors. A peptide substrate of activated transglutaminase factor XIII (FXIIIa)—a key ECM crosslinking enzyme—is rendered photosensitive by masking its active site with a photolabile cage group. Covalent incorporation of the caged FXIIIa substrate into poly(ethylene glycol) hydrogels and subsequent laser-scanning lithography affords highly localized biomolecule tethering. This approach for the 3D manipulation of cells within gels should open up avenues for the study and manipulation of cell signalling.

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Figure 1: Concept of light-controlled enzymatic biomolecule patterning of hydrogels.
Figure 2: Proof-of-principle of light-controlled enzymatic reactions using a soluble model system.
Figure 3: Spatial patterning of hydrogels with fluorescently labelled model ligands and proteins.
Figure 4: In situ manipulation of 3D MSC invasion by light-activated enzymatic patterning.

References

  1. Langer, R. & Tirrell, D. A. Designing materials for biology and medicine. Nature 428, 487–492 (2004).

    CAS  Article  Google Scholar 

  2. Lutolf, M. P. & Hubbell, J. A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nature Biotechnol. 23, 47–55 (2005).

    CAS  Article  Google Scholar 

  3. Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    CAS  Google Scholar 

  4. Lutolf, M. P., Doyonnas, R., Havenstrite, K., Koleckar, K. & Blau, H. M. Perturbation of single hematopoietic stem cell fates in artificial niches. Int. Biol. 1, 59–69 (2009).

    CAS  Google Scholar 

  5. Gilbert, P. M. et al. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science 329, 1078–1081 (2010).

    CAS  Article  Google Scholar 

  6. Lee, K., Silva, E. A. & Mooney, D. J. Growth factor delivery-based tissue engineering: General approaches and a review of recent developments. J. R. Soc. Inter. 8, 153–170 (2011).

    CAS  Article  Google Scholar 

  7. Katz, J. S. & Burdick, J. A. Light-responsive biomaterials: Development and applications. Macromol. Biosci. 10, 339–348 (2010).

    CAS  Article  Google Scholar 

  8. Kloxin, A., Kasko, A. M., Salinas, C. N. & Anseth, K. Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science 324, 59–63 (2009).

    CAS  Article  Google Scholar 

  9. Wong, D. Y., Griffin, D. R., Reed, J. & Kasko, A. M. Photodegradable hydrogels to generate positive and negative features over multiple length scales. Macromolecules 43, 2824–2831 (2010).

    CAS  Article  Google Scholar 

  10. Ramanan, V. V. et al. Photocleavable side groups to spatially alter hydrogel properties and cellular interactions. J. Mater. Chem. 20, 8920–8926 (2010).

    CAS  Article  Google Scholar 

  11. Luo, Y. & Shoichet, M. S. A photolabile hydrogel for guided three-dimensional cell growth and migration. Nature Mater. 3, 249–253 (2004).

    CAS  Article  Google Scholar 

  12. Hahn, M., Miller, J. & West, J. Three-dimensional biochemical and biomechanical patterning of hydrogels for guiding cell behavior. Adv. Mater. 18, 2679–2684 (2006).

    CAS  Article  Google Scholar 

  13. Wosnick, J. H. & Shoichet, M. S. Three-dimensional chemical patterning of transparent hydrogels. Chem. Mater. 20, 55–60 (2008).

    CAS  Article  Google Scholar 

  14. DeForest, C. A., Polizzotti, B. D. & Anseth, K. S. Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments. Nature Mater. 8, 659–664 (2009).

    CAS  Article  Google Scholar 

  15. Wylie, R. G. et al. Spatially controlled simultaneous patterning of multiple growth factors in three-dimensional hydrogels. Nature Mater. 10, 799–806 (2011).

    CAS  Article  Google Scholar 

  16. Ellis-Davies, G. C. R. Caged compounds: Photorelease technology for control of cellular chemistry and physiology. Nature Methods 4, 619–628 (2007).

    CAS  Article  Google Scholar 

  17. Lorand, L. & Graham, R. M. Transglutaminases: Crosslinking enzymes with pleiotropic functions. Nature Rev. Mol. Cell Biol. 4, 140–156 (2003).

    CAS  Article  Google Scholar 

  18. Mosesson, M. W., Siebenlist, K. R. & Meh, D. A. The structure and biological features of fibrinogen and fibrin. Fibrinogen 936, 11–30 (2001).

    CAS  Google Scholar 

  19. Sperinde, J. J. & Griffith, L. G. Synthesis and characterization of enzymatically-cross-linked poly(ethylene glycol) hydrogels. Macromolecules 30, 5255–5264 (1997).

    CAS  Article  Google Scholar 

  20. Hu, B. H. & Messersmith, P. B. Rational design of transglutaminase substrate peptides for rapid enzymatic formation of hydrogels. J. Am. Chem. Soc. 125, 14298–14299 (2003).

    CAS  Article  Google Scholar 

  21. Ehrbar, M. et al. Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering. Biomaterials 28, 3856–3866 (2007).

    CAS  Article  Google Scholar 

  22. Chan, B. P. Biomedical applications of photochemistry. Tissue Eng. B 16, 509–522 (2010).

    CAS  Article  Google Scholar 

  23. Lutolf, M. P. & Hubbell, J. A. Synthesis and physicochemical characterization of end-linked poly(ethylene glycol)-co-peptide hydrogels formed by Michael-type addition. Biomacromolecules 4, 713–722 (2003).

    CAS  Article  Google Scholar 

  24. Martino, M. M. et al. Controlling integrin specificity and stem cell differentiation in 2D and 3D environments through regulation of fibronectin domain stability. Biomaterials 30, 1089–1097 (2009).

    CAS  Article  Google Scholar 

  25. Ehrbar, M. et al. Cell-demanded liberation of VEGF(121) from fibrin implants induces local and controlled blood vessel growth. Circ. Res. 94, 1124–1132 (2004).

    CAS  Article  Google Scholar 

  26. Spaeth, E., Klopp, A., Dembinski, J., Andreeff, M. & Marini, F. Inflammation and tumor microenvironments: Defining the migratory itinerary of mesenchymal stem cells. Gene Ther. 15, 730–738 (2008).

    CAS  Article  Google Scholar 

  27. Martino, M. M. et al. Engineering the growth factor microenvironment with fibronectin domains to promote wound and bone tissue healing. Sci. Transl. Med. 3, 100ra89 (2011).

    Article  Google Scholar 

  28. Docheva, D., Popov, C., Mutschler, W. & Schieker, M. Human mesenchymal stem cells in contact with their environment: Surface characteristics and the integrin system. J. Cell Mol. Med. 11, 21–38 (2007).

    CAS  Article  Google Scholar 

  29. Tokunaga, A. et al. PDGF receptor beta is a potent regulator of mesenchymal stromal cell function. J. Bone Miner. Res. 23, 1519–1528 (2008).

    CAS  Article  Google Scholar 

  30. Veevers-Lowe, J., Ball, S. G., Shuttleworth, A. & Kielty, C. M. Mesenchymal stem cell migration is regulated by fibronectin through α5β1-integrin-mediated activation of PDGFR- β and potentiation of growth factor signals. J. Cell Sci. 124, 1288–1300 (2011).

    CAS  Article  Google Scholar 

  31. Ohmuro-Matsuyama, Y. & Tatsu, Y. Photocontrolled cell adhesion on a surface functionalized with a caged arginine-glycine-aspartate peptide. Angew. Chem. Int. Ed. 47, 7527–7529 (2008).

    CAS  Article  Google Scholar 

  32. Miller, D. S., Chirayil, S., Ball, H. L. & Luebke, K. J. Manipulating cell migration and proliferation with a light-activated polypeptide. ChemBioChem 10, 577–584 (2009).

    CAS  Article  Google Scholar 

  33. Rusiecki, V. K. & Warne, S. A. Synthesis of N-α-Fmoc-N-ɛ-Nvoc-lysine and use in the preparation of selectively functionalized peptides. Bioorg. Med. Chem. Lett. 3, 707–710 (1993).

    CAS  Article  Google Scholar 

  34. Cordey, M., Limacher, M., Kobel, S., Taylor, V. & Lutolf, M. P. Enhancing the reliability and throughput of neurosphere culture on hydrogel microwell arrays. Stem Cells 26, 2586–2594 (2008).

    Article  Google Scholar 

  35. Semenov, O. V. et al. Multipotent mesenchymal stem cells from human placenta: Critical parameters for isolation and maintenance of stemness after isolation. Am. J. Obstet. Gynecol. 202, 193.e1–193.e13 (2010).

    Article  Google Scholar 

  36. Korff, T. & Augustin, H. G. Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation. J. Cell. Biol. 143, 1341–1352 (1998).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank A. Negro for help with data analysis, A. Ranga, S. Allazetta, N. Brandenberg, Y. Okawa, K. Krishnamani, P. Abdel-Sayed and N. Balashubrmaniam for valuable discussion, C. Dessibourg, P. Briquez and the Protein Expression Core Facility of EPFL for assistance with recombinant protein production, A. Seitz and T. Laroche for support with confocal microscopy, R. Guiet and O. Burri for assistance with image processing, and S. Banala for sharing his experience with photocaging systems. This work was financially supported in part by a European Young Investigator (EURYI) Award (PE002-117115/1) and an ERC starting grant to M.P.L.

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M.P.L., K.A.M. and L.K. designed research, analysed data and wrote the paper; K.A.M. and L.K. performed research; A.J.v.d.V. contributed to synthesis, purification and analysis of caged molecules; M.M.M., P.S.L., J.A.H. and M.E. contributed new reagents/analytic tools. All authors gave input on the manuscript draft.

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Correspondence to Matthias P. Lutolf.

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Mosiewicz, K., Kolb, L., van der Vlies, A. et al. In situ cell manipulation through enzymatic hydrogel photopatterning. Nature Mater 12, 1072–1078 (2013). https://doi.org/10.1038/nmat3766

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