Stiffness-controlled three-dimensional extracellular matrices for high-resolution imaging of cell behavior

Regulation of cell functions by the physical properties of the extracellular matrix (ECM) has emerged as a crucial contributor to development and disease. Two specific physical properties of the ECM, stiffness and dimensionality, each influence cell signaling and function. As these ECM physical properties are linked to other properties that also regulate cell behavior, e.g., integrin ligand density, parsing the specific contributions of ECM stiffness and dimensionality has proven difficult. Here we detail a simple protocol, which can be completed in 1–2 d, for combining three-dimensional (3D) ECM engagement with controlled underlying ECM stiffness. In these 'sandwich gels', cells are sandwiched between a 3D fibrillar ECM and an ECM-coupled polyacrylamide gel of defined compliance, allowing the study of the specific effects of ECM compliance on cell function in physiologically relevant 3D ECMs. This type of system enables high-resolution time-lapse imaging and is suitable for a wide range of cell types and molecular perturbations.

[1]  D. Ingber,et al.  Prevascularization of porous biodegradable polymers , 1993, Biotechnology and bioengineering.

[2]  N. Shepherd,et al.  New relationships between breast microcalcifications and cancer , 2010, British Journal of Cancer.

[3]  N. Langrana,et al.  Effect of dynamic stiffness of the substrates on neurite outgrowth by using a DNA-crosslinked hydrogel. , 2010, Tissue engineering. Part A.

[4]  Daniel J. Maltman,et al.  Developments in three-dimensional cell culture technology aimed at improving the accuracy of in vitro analyses. , 2010, Biochemical Society transactions.

[5]  Yukinori Endo,et al.  A Rac switch regulates random versus directionally persistent cell migration , 2005, The Journal of cell biology.

[6]  R. Hard,et al.  Newt lung epithelial cells: cultivation, use, and advantages for biomedical research. , 1990, International review of cytology.

[7]  Dennis E Discher,et al.  Stem cells feel the difference , 2010, Nature Methods.

[8]  Dennis E Discher,et al.  How deeply cells feel: methods for thin gels , 2010, Journal of physics. Condensed matter : an Institute of Physics journal.

[9]  Mikala Egeblad,et al.  Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling , 2009, Cell.

[10]  Sanjay Kumar,et al.  Independent regulation of tumor cell migration by matrix stiffness and confinement , 2012, Proceedings of the National Academy of Sciences.

[11]  E. Cukierman,et al.  Physico-mechanical aspects of extracellular matrix influences on tumorigenic behaviors. , 2010, Seminars in cancer biology.

[12]  P. Janmey,et al.  Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. , 2005, Cell motility and the cytoskeleton.

[13]  Estuardo Robles,et al.  Focal adhesion kinase signaling at sites of integrin-mediated adhesion controls axon pathfinding , 2006, Nature Neuroscience.

[14]  D. Ingber,et al.  Rho signaling and mechanical control of vascular development , 2008, Current opinion in hematology.

[15]  D. Ingber,et al.  From 3D cell culture to organs-on-chips. , 2011, Trends in cell biology.

[16]  Dietmar W Hutmacher,et al.  Translating tissue engineering technology platforms into cancer research , 2009, Journal of cellular and molecular medicine.

[17]  S. Sen,et al.  Matrix Elasticity Directs Stem Cell Lineage Specification , 2006, Cell.

[18]  Y. Wang,et al.  Cell locomotion and focal adhesions are regulated by the mechanical properties of the substrate. , 1998, The Biological bulletin.

[19]  Donald E. Ingber,et al.  Mechanosensitive mechanisms in transcriptional regulation , 2012, Journal of Cell Science.

[20]  D. Ingber,et al.  Reconstituting Organ-Level Lung Functions on a Chip , 2010, Science.

[21]  Kristopher E Kubow,et al.  Reducing background fluorescence reveals adhesions in 3D matrices , 2012, Nature Cell Biology.

[22]  Mikala Egeblad,et al.  Dynamic interplay between the collagen scaffold and tumor evolution. , 2010, Current opinion in cell biology.

[23]  D. Ingber,et al.  Mechanotransduction at a distance: mechanically coupling the extracellular matrix with the nucleus , 2009, Nature Reviews Molecular Cell Biology.

[24]  D. Discher,et al.  Microscopic methods for measuring the elasticity of gel substrates for cell culture: microspheres, microindenters, and atomic force microscopy. , 2007, Methods in cell biology.

[25]  T. Gómez,et al.  Imaging adhesion and signaling dynamics in Xenopus laevis growth cones , 2012, Developmental neurobiology.

[26]  P. Wadsworth.  Using cultured mammalian cells to study mitosis. , 2012, Cold Spring Harbor protocols.

[27]  Stephanie I. Fraley,et al.  A distinctive role for focal adhesion proteins in three-dimensional cell motility , 2010, Nature Cell Biology.

[28]  Kristopher E Kubow,et al.  Reducing background fluorescence reveals adhesions in 3D matrices , 2012, Nature Cell Biology.

[29]  S. Inoué,et al.  Micromanipulation studies of the asymmetric positioning of the maturation spindle in Chaetopterus sp. oocytes: I. Anchorage of the spindle to the cortex and migration of a displaced spindle. , 1988, Cell motility and the cytoskeleton.

[30]  J. Girault,et al.  Organization of point contacts in neuronal growth cones , 1999, Journal of neuroscience research.

[31]  Joyce Y Wong,et al.  Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity , 2007, Journal of neural engineering.

[32]  M. Dembo,et al.  Cell movement is guided by the rigidity of the substrate. , 2000, Biophysical journal.

[33]  Kenneth M. Yamada,et al.  Direct comparisons of the morphology, migration, cell adhesions, and actin cytoskeleton of fibroblasts in four different three-dimensional extracellular matrices. , 2011, Tissue engineering. Part A.

[34]  D. Ingber,et al.  Mechanical behavior in living cells consistent with the tensegrity model , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[35]  P. Janmey,et al.  Tissue Cells Feel and Respond to the Stiffness of Their Substrate , 2005, Science.

[36]  B. Geiger,et al.  Environmental sensing through focal adhesions , 2009, Nature Reviews Molecular Cell Biology.

[37]  D. Ingber.  Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology. , 2002, Circulation research.

[38]  Daniel Gioeli,et al.  Matrix Rigidity Regulates Cancer Cell Growth and Cellular Phenotype , 2010, PloS one.

[39]  David J. Mooney,et al.  Non-viral gene delivery regulated by stiffness of cell adhesion substrates , 2005, Nature materials.

[40]  K. Beningo,et al.  Double-hydrogel substrate as a model system for three-dimensional cell culture. , 2007, Methods in molecular biology.

[41]  Ying Mei,et al.  Combinatorial Development of Biomaterials for Clonal Growth of Human Pluripotent Stem Cells , 2010, Nature materials.

[42]  T. Gómez,et al.  Focal Adhesion Kinase Promotes Integrin Adhesion Dynamics Necessary for Chemotropic Turning of Nerve Growth Cones , 2011, The Journal of Neuroscience.

[43]  D. Ingber,et al.  Tumor-derived endothelial cells exhibit aberrant Rho-mediated mechanosensing and abnormal angiogenesis in vitro , 2008, Proceedings of the National Academy of Sciences.

[44]  A. Bailey,et al.  quantitative determination of collagen cross-links. , 2009, Methods in molecular biology.

[45]  R. Tompkins,et al.  Effect of collagen gel configuration on the cytoskeleton in cultured rat hepatocytes. , 1993, Experimental cell research.

[46]  Ning Wang,et al.  Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II–dependent manner , 2010, The Journal of cell biology.

[47]  Y. Wang,et al.  Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells. , 1998, Methods in enzymology.

[48]  M L Yarmush,et al.  Hepatocytes in collagen sandwich: evidence for transcriptional and translational regulation , 1992, The Journal of cell biology.

[49]  K. Shakesheff,et al.  Tissue engineering in the development of replacement technologies. , 2012, Advances in experimental medicine and biology.

[50]  R. Tompkins,et al.  Hepatocyte function and extracellular matrix geometry: long‐term culture in a sandwich configuration , 1989, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[51]  J. Hubbell,et al.  Molecularly engineered PEG hydrogels: a novel model system for proteolytically mediated cell migration. , 2005, Biophysical journal.

[52]  Kenneth M. Yamada,et al.  Myosin IIA regulates cell motility and actomyosin–microtubule crosstalk , 2007, Nature Cell Biology.

[53]  Joe W. Gray,et al.  HER2 signaling pathway activation and response of breast cancer cells to HER2-targeting agents is dependent strongly on the 3D microenvironment , 2010, Breast Cancer Research and Treatment.

[54]  D E Ingber,et al.  Preparation of poly(glycolic acid) bonded fiber structures for cell attachment and transplantation. , 1993, Journal of biomedical materials research.

[55]  Donald E. Ingber,et al.  A mechanosensitive transcriptional mechanism that controls angiogenesis , 2009, Nature.

[56]  F. Grinnell,et al.  The differential regulation of cell motile activity through matrix stiffness and porosity in three dimensional collagen matrices. , 2010, Biomaterials.

[57]  C. M. Waterman-Storer,et al.  Microtubule/Organelle Motility Assays , 1998, Current protocols in cell biology.

[58]  James B Phillips,et al.  Neural tissue engineering: a self-organizing collagen guidance conduit. , 2005, Tissue engineering.

[59]  D. Slatter,et al.  Collagen in its fibrillar state is protected from glycation. , 2008, The international journal of biochemistry & cell biology.

[60]  Frederik De Smet,et al.  Branching morphogenesis and antiangiogenesis candidates: tip cells lead the way , 2009, Nature Reviews Clinical Oncology.

[61]  Florian Rehfeldt,et al.  Hyaluronic acid matrices show matrix stiffness in 2D and 3D dictates cytoskeletal order and myosin-II phosphorylation within stem cells. , 2012, Integrative biology : quantitative biosciences from nano to macro.

[62]  T. Gómez,et al.  Regulation of axonal outgrowth and pathfinding by integrin–ecm interactions , 2011, Developmental neurobiology.

[63]  Robert S. Adelstein,et al.  Local Cortical Tension by Myosin II Guides 3D Endothelial Cell Branching , 2009, Current Biology.

[64]  Yuda Fang,et al.  Live cell imaging of plants. , 2010, Cold Spring Harbor protocols.

[65]  K. Beningo,et al.  Responses of fibroblasts to anchorage of dorsal extracellular matrix receptors , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[66]  Kenneth M. Yamada,et al.  Random versus directionally persistent cell migration , 2009, Nature Reviews Molecular Cell Biology.

[67]  Valerie M. Weaver,et al.  Three-dimensional context regulation of metastasis , 2008, Clinical & Experimental Metastasis.

[68]  Dihua Yu,et al.  Cancer cell stiffness: integrated roles of three-dimensional matrix stiffness and transforming potential. , 2010, Biophysical journal.

[69]  Kinam Park,et al.  Advances in superporous hydrogels. , 2005, Journal of controlled release : official journal of the Controlled Release Society.

[70]  Gaudenz Danuser,et al.  Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis , 2011, The Journal of cell biology.

[71]  Kenneth M. Yamada,et al.  Cell-matrix adhesions in 3D. , 2011, Matrix biology : journal of the International Society for Matrix Biology.

[72]  Kenneth M. Yamada,et al.  One-dimensional topography underlies three-dimensional fibrillar cell migration , 2009, The Journal of cell biology.

[73]  D. G. T. Strange,et al.  Extracellular-matrix tethering regulates stem-cell fate. , 2012, Nature materials.

[74]  Marion Ghibaudo,et al.  Rigidity-driven growth and migration of epithelial cells on microstructured anisotropic substrates , 2007, Proceedings of the National Academy of Sciences.

[75]  B. Olsen.  Life without Perlecan Has Its Problems , 1999, The Journal of cell biology.

[76]  Frederick Grinnell,et al.  Cell motility and mechanics in three-dimensional collagen matrices. , 2010, Annual review of cell and developmental biology.