There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 μm to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.
20 Related JoVE Articles!
Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium
Institutions: Duke University , University of Rochester , University of Rochester .
The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity.
Microcontact printing (μ
CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9
Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 μ
In contrast to traditional printing, inkless μ
CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (<200 nm) features.17-23
However, up till now, inkless μ
CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many μ
CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.
Bioengineering, Issue 58, Soft lithography, microcontact printing, protein arrays, catalytic printing, oxide-free silicon
Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement
Institutions: Washington University in St. Louis.
Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1
While microcontact printing can be employed to directly print a large number of molecules, including proteins,2
the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5
This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6
These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7
to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9
patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10
Bioengineering, Issue 55, Self-assembled monolayer (SAM), microcontact printing, protein patterning, patterned cell growth
Silk Film Culture System for in vitro Analysis and Biomaterial Design
Institutions: Weill Cornell Medical College , Tufts University.
Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2
. These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8
. In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro
or in vivo
, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13
. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14
. The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro
culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo
models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.
Bioengineering, Issue 62, silk, fibroin, film, biomaterial, surface patterning, in vitro, epithelium, cell culture
Plasma Lithography Surface Patterning for Creation of Cell Networks
Institutions: University of Arizona , University of Arizona .
Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.
Bioengineering, Issue 52, Cell Network, Surface Patterning, Self-Organization, Developmental Biology, Tissue Engineering, Nanopattern, Micropattern, Self-Assembly, Cell Guidance, Neuron
A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research
Institutions: Arizona State University.
Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g.
, food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera
) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.
Neuroscience, Issue 91, PER, conditioning, honey bee, olfaction, olfactory processing, learning, memory, toxin assay
Fabrication of Uniform Nanoscale Cavities via Silicon Direct Wafer Bonding
Institutions: The State University of New York at Buffalo, University of Maryland, The National Institute of Standards and Technology, NASA Goddard Space Flight Center, HRL Laboratories.
Measurements of the heat capacity and superfluid fraction of confined 4
He have been performed near the lambda transition using lithographically patterned and bonded silicon wafers. Unlike confinements in porous materials often used for these types of experiments3
, bonded wafers provide predesigned uniform spaces for confinement. The geometry of each cell is well known, which removes a large source of ambiguity in the interpretation of data.
Exceptionally flat, 5 cm diameter, 375 µm thick Si wafers with about 1 µm variation over the entire wafer can be obtained commercially (from Semiconductor Processing Company, for example). Thermal oxide is grown on the wafers to define the confinement dimension in the z-direction. A pattern is then etched in the oxide using lithographic techniques so as to create a desired enclosure upon bonding. A hole is drilled in one of the wafers (the top) to allow for the introduction of the liquid to be measured. The wafers are cleaned2
in RCA solutions and then put in a microclean chamber where they are rinsed with deionized water4
. The wafers are bonded at RT and then annealed at ~1,100 °C. This forms a strong and permanent bond. This process can be used to make uniform enclosures for measuring thermal and hydrodynamic properties of confined liquids from the nanometer to the micrometer scale.
Physics, Issue 83, silicon direct wafer bonding, nanoscale, bonded wafers, silicon wafer, confined liquids, lithographic techniques
An Improved Mechanical Testing Method to Assess Bone-implant Anchorage
Institutions: University of Toronto.
Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces - namely numerical determinants of surface roughness - are inadequate for predicting in vivo
performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces - but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.
Bioengineering, Issue 84, Mechanical test, bone anchorage, disruption test, surface topography, peri-implant bone, bone-implant interface, bone-bonding, microtopography, nanotopography
Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies
Institutions: University of California, Davis , University of California, Davis , University of California, Davis .
Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.
Bioengineering, Issue 77, Cellular Biology, Molecular Biology, Biomedical Engineering, Biochemistry, Chemistry, Chemical Engineering, Biophysics, Physics, Nanotechnology, Nanostructures, Biomedical Technology, Miniaturization, Gold, Staining and Labeling, Cell Culture Techniques, Microscopy, Electron Microscopy, Fluorescence, Nanotechnology, thin films (theory, deposition and growth), Nanoporous gold, cell culture, image analysis, microfabrication, nanotechnology, quantitative immunochemistry, scanning electron microscopy, SEM, fluorescence microscopy, stencil printing, photolithography, cell culture
Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns
Institutions: MIT - Massachusetts Institute of Technology, MIT - Massachusetts Institute of Technology, Brigham and Women's Hospital and Harvard Medical School.
Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1
. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2
. Well-defined, μm-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3
were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1
. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.
Bioengineering, Issue 48, cell rolling, microcontact printing, cell adhesion, cell analysis, cell separation, P-selectin
Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers
Institutions: University of Maryland, University of Maryland.
Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200 nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125
I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125
I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.
Bioengineering, Issue 80, Antigens, Enzymes, Biological Therapy, bioengineering (general), Pharmaceutical Preparations, Macromolecular Substances, Therapeutics, Digestive System and Oral Physiological Phenomena, Biological Phenomena, Cell Physiological Phenomena, drug delivery systems, targeted nanocarriers, transcellular transport, epithelial cells, tight junctions, transepithelial electrical resistance, endocytosis, transcytosis, radioisotope tracing, immunostaining
Characterization of Surface Modifications by White Light Interferometry: Applications in Ion Sputtering, Laser Ablation, and Tribology Experiments
Institutions: Argonne National Laboratory, Argonne National Laboratory, MassThink LLC.
In materials science and engineering it is often necessary to obtain quantitative measurements of surface topography with micrometer lateral resolution. From the measured surface, 3D topographic maps can be subsequently analyzed using a variety of software packages to extract the information that is needed.
In this article we describe how white light interferometry, and optical profilometry (OP) in general, combined with generic surface analysis software, can be used for materials science and engineering tasks. In this article, a number of applications of white light interferometry for investigation of surface modifications in mass spectrometry, and wear phenomena in tribology and lubrication are demonstrated. We characterize the products of the interaction of semiconductors and metals with energetic ions (sputtering), and laser irradiation (ablation), as well as ex situ
measurements of wear of tribological test specimens.
Specifically, we will discuss:
Aspects of traditional ion sputtering-based mass spectrometry such as sputtering rates/yields measurements on Si and Cu and subsequent time-to-depth conversion.
Results of quantitative characterization of the interaction of femtosecond laser irradiation with a semiconductor surface. These results are important for applications such as ablation mass spectrometry, where the quantities of evaporated material can be studied and controlled via pulse duration and energy per pulse. Thus, by determining the crater geometry one can define depth and lateral resolution versus experimental setup conditions.
Measurements of surface roughness parameters in two dimensions, and quantitative measurements of the surface wear that occur as a result of friction and wear tests.
Some inherent drawbacks, possible artifacts, and uncertainty assessments of the white light interferometry approach will be discussed and explained.
Materials Science, Issue 72, Physics, Ion Beams (nuclear interactions), Light Reflection, Optical Properties, Semiconductor Materials, White Light Interferometry, Ion Sputtering, Laser Ablation, Femtosecond Lasers, Depth Profiling, Time-of-flight Mass Spectrometry, Tribology, Wear Analysis, Optical Profilometry, wear, friction, atomic force microscopy, AFM, scanning electron microscopy, SEM, imaging, visualization
Capillary Force Lithography for Cardiac Tissue Engineering
Institutions: University of Washington, University of Washington.
Cardiovascular disease remains the leading cause of death worldwide1
. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro
screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart’s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2
. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2
. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo
structural and substrate stiffness cues of the ECM in the heart6-9
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8
and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16
. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (λ = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 °C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2
Bioengineering, Issue 88, Nanotopography, Anisotropic, Nanofabrication, Cell Culture, Cardiac Tissue Engineering
Cell Patterning on Photolithographically Defined
Parylene-C: SiO2 Substrates
Institutions: The University of Edinburgh, Western General Hospital, The University of Edinburgh.
Cell patterning platforms support broad research goals, such as construction of predefined in vitro
neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based ‘lab on a chip’ technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2
wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2
regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.
Bioengineering, Issue 85, Receptors, Cell Surface, Polymers, Cell Adhesion, Biomedical and Dental Materials, parylene-C, silicon dioxide, photolithography, cell adhesion, Cell Patterning
Cell Co-culture Patterning Using Aqueous Two-phase Systems
Institutions: University of Michigan , University of Michigan .
Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.
Bioengineering, Issue 73, 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
In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions
Institutions: Pacific Northwest National Laboratory.
Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ
characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3
(bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ
time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ
Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ
IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.
Chemistry, Issue 88, soft landing, mass selected ions, electrospray, secondary ion mass spectrometry, infrared spectroscopy, organometallic, catalysis
Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-µCP) and Sequential Nucleophilic Substitution
Institutions: University of Wisconsin, Madison, University of Wisconsin, Madison.
In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo
niches. Microcontact printing (μCP) is a versatile technique to pattern such grafted PEG brushes, but manual μCP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-μCP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal ‘click’ chemistries. Here, we describe in detail the workflow to manufacture such substrates.
Bioengineering, Issue 92, Robotic microcontact printing, R-μCP, click chemistry, surface chemistry, tissue engineering, micropattern, advanced manufacturing
Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues
Institutions: Université de Mons.
It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro
. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro
cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.
Bioengineering, Issue 90, hydrogels, mechanotransduction, polyacrylamide, microcontact printing, cell shape, stiffness, durotaxis, cell-ligand density
Microcontact Printing of Proteins for Cell Biology
Institutions: Columbia University.
The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (https://gmwgroup.harvard.edu/) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1
. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2
. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.
Cellular Biology, Issue 22, micropatterning, proteins, cell biology, microcontact
Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes
Institutions: University of California, Davis.
The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues. In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates. The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive. Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule. Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks. Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns. Micropatterning of several cell types was demonstrated on these substrates. In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.
Cellular Biology, Issue 7, indium tin oxide, surface modification, electrochemistry, cell patterning
A Microfluidic Device with Groove Patterns for Studying Cellular Behavior
Institutions: Brigham and Women's Hospital.
We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 μm wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37°C, 5% CO2
). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.
Issue 7, Cell Biology, tissue engineering, microfluidic, apoptosis