Probing cellular mechanobiology in three-dimensional culture with collagen– agarose matrices Theresa A. Ulrich a, b , Amit Jain a, 1 , Kandice Tanner a , Joanna L. MacKay a, c , Sanjay Kumar a, b, * a Department of Bioengineering, University of California, Berkeley, CA 94720-1762, USA b University of California, San Francisco/University of California, Berkeley Joint Graduate Group in Bioengineering, Berkeley, CA 94720, USA c Department of Chemical Engineering, University of California, Berkeley, CA 94720, USA article info Article history: Received 28 September 2009 Accepted 20 October 2009 Available online 18 November 2009 Keywords: Hydrogel Collagen ECM (extracellular matrix) Mechanical properties Elasticity Brain abstract The study of how cell behavior is controlled by the biophysical properties of the extracellular matrix (ECM) is limited in part by the lack of three-dimensional (3D) scaffolds that combine the biofunctionality of native ECM proteins with the tunability of synthetic materials. Here, we introduce a biomaterial platform in which the biophysical properties of collagen I are progressively altered by adding agarose. We find that agarose increases the elasticity of 3D collagen ECMs over two orders of magnitude with modest effect on collagen fiber organization. Surprisingly, increasing the agarose content slows and eventually stops invasion of glioma cells in a 3D spheroid model. Electron microscopy reveals that agarose forms a dense meshwork between the collagen fibers, which we postulate slows invasion by structurally coupling and reinforcing the collagen fibers and introducing steric barriers to motility. This is supported by time lapse imaging of individual glioma cells and multicellular spheroids, which shows that addition of agarose promotes amoeboid motility and restricts cell-mediated remodeling of individual collagen fibers. Our results are consistent with a model in which agarose shifts ECM dissipation of cell-induced stresses from non-affine deformation of individual collagen fibers to bulk-affine deformation of a continuum network. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction The behavior of many mammalian cell types is exquisitely sensitive to biophysical signals from the extracellular matrix (ECM), including those encoded within matrix mechanical properties [1]. The robust mechanosensitivity of mammalian cells has been extensively probed in vitro using two-dimensional (2D) biomaterial platforms that feature full-length ECM proteins covalently conju- gated to polymeric hydrogels of defined stiffness [2], thus allowing independent modulation of ECM mechanical and biochemical properties. We and others have used these platforms to demon- strate that cellular structure, motility, and proliferation can be strongly regulated by the mechanical rigidity of the ECM [2,3]. However, many cells reside in a three-dimensional (3D) ECM in vivo, and it is now well-established that culture dimensionality strongly impacts gene expression, cell adhesion and migration, and assembly into multicellular structures [4,5]. Although there is increasing interest in investigating cellular mechanobiological properties in 3D culture systems, the develop- ment of matrices for this purpose has proven challenging. One common strategy is based on incorporation of cell adhesion peptides (e.g., RGD) into synthetic polymer networks [6–8]; while this frequently offers robust and independent control of ECM ligand density and mechanics, it necessarily sacrifices the rich biochemical and topological information encoded in networks of full-length matrix proteins. Conversely, strategies that control 3D matrix properties by varying the concentration of a native ECM formula- tion (e.g., collagen, Matrigel) access a fairly narrow stiffness range due to the intrinsically low elasticity of most of these materials and concurrently change ECM mechanics, ligand density, microstruc- ture, and other potentially confounding biophysical parameters [9,10]. Collagen I is both the most abundant ECM protein in mamma- lian tissues and one of the most widely-used scaffolds for three- dimensional cell culture and tissue engineering applications, which is partly derived from the ability of purified collagen I monomers to self-assemble into stable, three-dimensional gels at physiological * Corresponding author. Department of Bioengineering, University of California, Berkeley, CA 94720-1762, USA. Tel.: þ1 510 643 0787; fax: þ1 510 642 5835. E-mail address: skumar@berkeley.edu (S. Kumar). 1 Present address: Amit Jain’s present address is Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.10.047 Biomaterials 31 (2010) 1875–1884