1Department of Biomedical Engineering, University of Wisconsin, Madison, 2Department of Mechanical Engineering, University of Wisconsin, Madison
Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.
Published October 31, 2014. Keywords: Bioengineering, Robotic microcontact printing, R-μCP, click chemistry, surface chemistry, tissue engineering, micropattern, advanced manufacturing
1Cardiovascular Research Center, Massachusetts General Hospital and Harvard Medical School, 2Harvard Stem Cell Institute
The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.
Published March 19, 2013. Keywords: Stem Cell Biology, Bioengineering, Biomedical Engineering, Medicine, Molecular Biology, Cellular Biology, Anatomy, Physiology, Tissue Engineering, Cardiology, Cell Biology, Embryonic Stem Cells, ESCs, Micropatterning, Microcontact Printing, Cell Alignment, Heart Progenitors, in vitro Differentiation, Transgenic Mice, Mouse Embryonic Stem Cells, stem cells, myocardial tissue, PDMS, FACS, flow cytometry, animal model
1Department of Biomedical Engineering, Columbia University
Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.
Published December 5, 2008. Keywords: Cellular Biology, micropatterning, proteins, cell biology, microcontact
1Centre for Vascular Research and Australian Centre for Nanomedicine, The University of New South Wales, 2School of Chemistry and Australian Centre for Nanomedicine, The University of New South Wales
A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.
Published April 4, 2013. Keywords: Bioengineering, Microbiology, Cellular Biology, Biochemistry, Molecular Biology, Biophysics, Cell migration, live cell imaging, soluble and adherent gradients, microcontact printing, dip pen lithography, microfluidics, RGD, PEG, biotin, streptavidin, chemotaxis, chemoattractant, imaging
1Department of Chemistry, Washington University in St. Louis
Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.
Published September 6, 2011. Keywords: Bioengineering, Self-assembled monolayer (SAM), microcontact printing, protein patterning, patterned cell growth
1Department of Chemistry, Duke University, 2Hajim School of Engineering and Applied Sciences, University of Rochester, 3Department of Chemical Engineering, University of Rochester
Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.
Published December 16, 2011. Keywords: Bioengineering, Soft lithography, microcontact printing, protein arrays, catalytic printing, oxide-free silicon
1Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland
In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.
Published October 15, 2013. Keywords: Immunology, Microfluidics, proteomics, systems biology, single-cell analysis, Immunoassays, Lab on a chip, chemical analysis
1Department of Biomedical Engineering, Carnegie Mellon University, 2Department of Materials Science and Engineering, Carnegie Mellon University
A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.
Published April 17, 2014. Keywords: Bioengineering, Nanofibers, Nanofabrics, Extracellular Matrix Proteins, Microcontact Printing, Fibronectin, Laminin, Tissue Engineering, poly(N-isopropylacrylamide), Surface-Initiated Assembly
1Laboratoire Interfaces et Fluides Complexes, Université de Mons
We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.
Published August 28, 2014. Keywords: Bioengineering, hydrogels, mechanotransduction, polyacrylamide, microcontact printing, cell shape, stiffness, durotaxis, cell-ligand density
1Department of Materials Science and Engineering, MIT - Massachusetts Institute of Technology, 2Department of Mechanical Engineering, MIT - Massachusetts Institute of Technology, 3HST Center for Biomedical Engineering and Harvard Stem Cell Institute, Brigham and Women's Hospital and Harvard Medical School
We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.
Published February 13, 2011. Keywords: Bioengineering, cell rolling, microcontact printing, cell adhesion, cell analysis, cell separation, P-selectin
1Department of Chemical and Biomolecular Engineering, Johns Hopkins Physical Sciences Oncology Center and Institute for NanoBioTechnology, Johns Hopkins University
A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.
Published December 22, 2010. Keywords: Bioengineering, Hyaluronic acid, microcontact printing, carbodiimide chemistry, cancer, cell adhesion
1Department of Bioengineering, University of Washington, 2Department of Pathology, University of Washington
In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.
Published June 10, 2014. Keywords: Bioengineering, Nanotopography, Anisotropic, Nanofabrication, Cell Culture, Cardiac Tissue Engineering
1Centre for Integrative Physiology, School of Biomedical Sciences, The University of Edinburgh, 2Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, Western General Hospital, 3School of Engineering, Institute for Integrated Micro and Nano Systems, The University of Edinburgh
This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.
Published March 7, 2014. Keywords: Bioengineering, Receptors, Cell Surface, Polymers, Cell Adhesion, Biomedical and Dental Materials, parylene-C, silicon dioxide, photolithography, cell adhesion, Cell Patterning
1Department of Biomedical Engineering, University of Michigan, 2Department of Macromolecular Science and Engineering, University of Michigan
Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.
Published March 26, 2013. Keywords: Bioengineering, Biomedical Engineering, Microbiology, Molecular Biology, Cellular Biology, Biochemistry, Biotechnology, Cell Migration Assays, Culture Techniques, bioengineering (general), Patterning, Aqueous Two-Phase System, Co-Culture, cell, Dextran, Polyethylene glycol, media, PEG, DEX, colonies, cell culture
1Department of Electrical and Computer Engineering, University of Minnesota, 2Department of Biomedical Engineering, University of Minnesota, 3Department of Neurology, Mayo Clinic College of Medicine, 4Department of Immunology, Mayo Clinic College of Medicine
Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.
Published May 8, 2014. Keywords: Bioengineering, supported lipid bilayer, beads, microarray, fluorescence, microfabrication, nanofabrication, atomic layer deposition, myelin, lipid rafts
1Center for Neuroscience, Swammerdam Institute for Life Sciences, University of Amsterdam, 2Van Leeuwenhoek Centre for Advanced Microscopy, Section Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam
This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.
Published May 4, 2014. Keywords: Neuroscience, Dendritic Spine, Microscopy, Confocal, Fluorescence, Neurosciences, hippocampus, primary neuron, super resolution microscopy, structured illumination microscopy (SIM), neuroscience, dendrite
JoVE Application Notes
In vitro mammalian cell culture has served as an invaluable tool in cell biology for several decades. Classically, monolayer cultures of adherent cells were grown on flat and rigid two-dimensional (2D) substrates, such as polystyrene or glass. However, many cells, when isolated from tissues and placed onto stiff planar 2D cell culture surfaces, such as tissue culture plastic, become progressively flatter, divide aberrantly, and lose their differentiated phenotype1,2. While these two-dimensional cell culture studies have played a pivotal role in furthering our understanding of many biological processes, they do not emulate in vivo conditions.
Published October 16, 2014. Keywords:
1School of Biological Sciences, Nanyang Technological University
We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.
Published June 7, 2014. Keywords: Bioengineering, mica, bilayer, lipids, TIRFM, imaging, SMT, AFM
1Department of Biomedical Engineering, Cornell University, 2BioCytics, Inc., 3Carolina BioOncology Institute, PLLC
Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.
Published June 15, 2012. Keywords: Bioengineering, Biomedical Engineering, Cancer Biology, Circulating tumor cells, metastasis, selectin, nanotechnology, halloysite nanotubes, cell isolation, cancer
1Dept of Physics, MIT - Massachusetts Institute of Technology, 2Department of Electrical Engineering and Computer Science, MIT - Massachusetts Institute of Technology
We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.
Published October 1, 2007. Keywords: Cellular Biology, tissue engineering, stem cells, patterning, bioengineering, signaling, diffusible, autocrine, juxtacrine
1Institute of Biophysics, Johannes Kepler Universitat Linz
This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.
Published March 19, 2010. Keywords: Bioengineering, protein-protein interactions, quantification, in-vivo, micro-contact-printing, micro-patterned surfaces