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.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Communications Materials Open Access 14 February 2022
Communications Biology Open Access 10 December 2021
Scientific Reports Open Access 31 March 2020
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Langer, R. & Tirrell, D. A. Designing materials for biology and medicine. Nature 428, 487–492 (2004).
Lutolf, M. P. & Hubbell, J. A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nature Biotechnol. 23, 47–55 (2005).
Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).
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).
Gilbert, P. M. et al. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science 329, 1078–1081 (2010).
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).
Katz, J. S. & Burdick, J. A. Light-responsive biomaterials: Development and applications. Macromol. Biosci. 10, 339–348 (2010).
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).
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).
Ramanan, V. V. et al. Photocleavable side groups to spatially alter hydrogel properties and cellular interactions. J. Mater. Chem. 20, 8920–8926 (2010).
Luo, Y. & Shoichet, M. S. A photolabile hydrogel for guided three-dimensional cell growth and migration. Nature Mater. 3, 249–253 (2004).
Hahn, M., Miller, J. & West, J. Three-dimensional biochemical and biomechanical patterning of hydrogels for guiding cell behavior. Adv. Mater. 18, 2679–2684 (2006).
Wosnick, J. H. & Shoichet, M. S. Three-dimensional chemical patterning of transparent hydrogels. Chem. Mater. 20, 55–60 (2008).
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).
Wylie, R. G. et al. Spatially controlled simultaneous patterning of multiple growth factors in three-dimensional hydrogels. Nature Mater. 10, 799–806 (2011).
Ellis-Davies, G. C. R. Caged compounds: Photorelease technology for control of cellular chemistry and physiology. Nature Methods 4, 619–628 (2007).
Lorand, L. & Graham, R. M. Transglutaminases: Crosslinking enzymes with pleiotropic functions. Nature Rev. Mol. Cell Biol. 4, 140–156 (2003).
Mosesson, M. W., Siebenlist, K. R. & Meh, D. A. The structure and biological features of fibrinogen and fibrin. Fibrinogen 936, 11–30 (2001).
Sperinde, J. J. & Griffith, L. G. Synthesis and characterization of enzymatically-cross-linked poly(ethylene glycol) hydrogels. Macromolecules 30, 5255–5264 (1997).
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).
Ehrbar, M. et al. Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering. Biomaterials 28, 3856–3866 (2007).
Chan, B. P. Biomedical applications of photochemistry. Tissue Eng. B 16, 509–522 (2010).
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).
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).
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).
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).
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).
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).
Tokunaga, A. et al. PDGF receptor beta is a potent regulator of mesenchymal stromal cell function. J. Bone Miner. Res. 23, 1519–1528 (2008).
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).
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).
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).
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).
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).
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).
Korff, T. & Augustin, H. G. Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation. J. Cell. Biol. 143, 1341–1352 (1998).
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.
The authors declare no competing financial interests.
About this article
Cite this article
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
Communications Materials (2022)
Self-sorting double network hydrogels with photo-definable biochemical cues as artificial synthetic extracellular matrix
Nano Research (2022)
Nature Reviews Materials (2021)
Communications Biology (2021)
Scientific Reports (2020)