Micropatterning Technique Research Articles

Morphological Transformation of a Liquid Micropattern on Dynamically Tunable Microwrinkles

When a very thin, hard layer supported by a soft substrate is laterally compressed beyond a critical strain, then buckling of the hard layer occurs, leading to the formation of small sinusoidal surface undulations (microwrinkles). The orientation of the wrinkle grooves can be reversibly altered by simply adding a strain externally. Using this nonlinear microtopological change, we demonstrate that the morphology of an array of liquid filaments formed on microwrinkle grooves is dramatically and reversibly transformed into liquid filaments with a different orientation or a regular array of microdroplets, "dots", depending on the predefined contact angle. The novel liquid transformation at nanomicrometer scales will find unique applications, such as switchable light diffraction grating, laboratories-on-a-chip systems as well as a simple liquid micropatterning technique.

Spatially Controlled Oxygen Inhibition of Acrylate Photopolymerization as a New Lithography Method for High-Performance Organic Thin-Film Transistors

A new concept-based lithography with use of spatially controlled oxygen inhibition of photopolymerization in acrylate-based materials as a micropatterning technique and its application in high-performance top- and bottom-gated organic thin-film transistors (OTFTs) on flexible substrates are demonstrated. This process does not rely on any surface/interfacial chemistry or adhesives and thus can be used in the patterning of a broad range of materials for printed organic electronics. It is also capable of achieving high resolution (3 μm), which is sufficient in OTFT-based organic electronics applications. The bottom-gated pentacene transistors show a remarkably high mobility of 1.02 cm2 V−1 S−1, which is among the highest mobilities in flexible transistors fabricated by low-cost patterning processes.

Three-dimensional photolithographic patterning of multiple bioactive ligands in poly(ethylene glycol) hydrogels

The biomaterials community is faced with the challenge of imitating a vastly complex, physiological tissue environment. While cellular systems towards this end have been traditionally studied in two dimensions (2D), most cells require three-dimensional (3D) cues to produce a physiologically relevant response. Two-photon absorption laser scanning lithography (TPA-LSL) may be applied to photosensitive hydrogel systems to engineer heterogeneous, 3D microenvironments consisting of precisely patterned bioactive signals. In this work, we have developed new operating parameters and system capabilities for TPA-LSL through the patterning of fluorescently labeled monoacrylate PEG-RGDS within PEG-DA hydrogels. Specifically, we have demonstrated a flexible pattern size range, with features ranging from 1 µm to nearly 1 mm. We have also shown patterns of differing concentrations of the cell adhesive ligand RGDS and correlated observed RGDS fluorescence with laser scan speed and intensity. Finally, we have micropatterned multiple, unique bioactive ligands into distinct, 3D forms within a single hydrogel. The results presented here have significantly developed the capabilities of the TPA-LSL micropatterning technique to allow for the fabrication of heterogeneous, 3D cellular microenvironments, which should prove highly useful for future biomimetic applications.

BioSpotlight

BioSpotlight

Surface micropatterning technique for surface-enhanced Raman scattering analysis

A facile and powerful method to form micropatterns, based on nanosphere lithography and layer-by-layer self-assembly, is described in this paper. A monolayer of hexagon close-packed polystyrene (PS) spheres (av 600 nm) was employed as the template. A positively charged polyelectrolyte of poly(diallydimethylammonium) chloride was used as adhesive to absorb oppositely charged spherical Au (av 17 nm) and spherical Ag (av 12 nm), respectively. Both the PS and substrates were coated by colloidal nanoparticles. After removal of the PS template, a micropatterning surface was obtained. Scanning electron microscopy and atomic force microscopy were used to characterize the morphologies. These resulting patterns can be used as good surface-enhanced Raman scattering (SERS) substrates. The enhancement factor is calculated to be 104 ∼ 105. This is a simple and facile technique for surface modification of metal micropatterns with SERS analytical application.

A simple lift-off-based patterning method for micro- and nanostructuring of functional substrates for cell culture

The ability to produce cell patterning through precise surface engineering has stimulated the development of cellular bioassays that offer new insights on the mechanisms of cell adhesion, proliferation, differentiation and molecular signaling pathways. Here we describe a simple micropatterning technique combining supersonic cluster beam deposition of nanostructured titania films on bovine serum albumin functionalized substrates. A standard lift-off process enables us to generate complementary micropatterns of hydrophobic bovine serum albumin (cell-repellent) and hydrophilic nanostructured TiOx (cell-adhesive). We demonstrate the selective PC12 cell adhesion and growth on biocompatible nanostructured TiOx. We also observed that these functional micropatterned substrates promote a considerable enhancement of cell attachment and proliferation.

One-dimensional topography underlies three-dimensional fibrillar cell migration

Current concepts of cell migration were established in regular two-dimensional (2D) cell culture, but the roles of topography are poorly understood for cells migrating in an oriented 3D fibrillar extracellular matrix (ECM). We use a novel micropatterning technique termed microphotopatterning (μPP) to identify functions for 1D fibrillar patterns in 3D cell migration. In striking contrast to 2D, cell migration in both 1D and 3D is rapid, uniaxial, independent of ECM ligand density, and dependent on myosin II contractility and microtubules (MTs). 1D and 3D migration are also characterized by an anterior MT bundle with a posterior centrosome. We propose that cells migrate rapidly through 3D fibrillar matrices by a 1D migratory mechanism not mimicked by 2D matrices.

Surface modification by 2-methacryloyloxyethyl phosphorylcholine coupled to a photolabile linker for cell micropatterning

This report describes a new surface-treatment technique for cell micropatterning. Cell attachment was selectively controlled on the glass surface using a photochemical reaction. This strategy is based on combining 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, which is known to reduce non-specific adsorption, and a photolabile linker (PL) for selective cell patterning. The MPC polymer was coated directly on the glass surface using a straightforward surface modification method, and was removed by ultraviolet (UV) light illumination. All the surface modification steps were evaluated using static water contact angle measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), measurements of non-specific protein adsorption, and the cell attachment test. After selective cleavage of the MPC polymer through the photomask, cells attached only to the UV-illuminated region where the MPC polymer was removed, which made the hydrophilic surface relatively hydrophobic. Furthermore, the size of the MC-3T3 E1 cell patterns could be controlled by single cell level. Stability of the cell micropatterns was demonstrated by culturing MC-3T3 E1 cell patterns for 5 weeks on glass slide. The micropatterns were stable during culturing; cell viability also was verified. This method can be a powerful tool for cell patterning research.

Micropatterned organoid culture of rat hepatocytes and HepG2 cells

The culture of liver cell organoids (multicellular aggregates) such as spheroids or cylindroids, which can strongly express liver functions, has been advocated as a useful technique that has advantages over monolayer culture. This paper describes a micropatterning technique for obtaining spheroids and cylindroids by using rat hepatocytes or HepG2 cells. We developed culture chips that comprised multiple, circular or rectangular microwells; the bottom surface of each microwell was modified with collagen to create a cell adhesion area, and the entire microwell, excluding the collagen-coated spots, was modified with polyethylene glycol (PEG) to create a nonadhesive area. Rat hepatocytes and HepG2 cells formed uniform spheroids and cylindroids on the circular and rectangular chips, respectively. Consequently, two-dimensional micropatterned chips containing homogeneous spheroids or cylindroids were generated. The expression of liver functions (protein secretion and ammonia removal) was greater in the spheroids and cylindroids than in the monolayer culture, and this expression was maintained for at least 2 weeks of culture. Thus, this chip technology has potential for use in various applications that involve organoid culture.

Fabrication of passive glass micromixer with third-dimensional feature by employing SU8 mask on micro-abrasive jet machining

Micro-abrasive jet machining (μ-AJM) is a fast and flexible technique for micro-patterning of brittle materials and can be combined with patterned mask made from a material with excellent photolithographic properties. Here, we demonstrate a fabrication method for the realization of a passive micromixer with third-dimensional feature by using a μ-AJM process with employing photopolymer as a mask on a glass slide target. We fabricated the mask using SU8, a photosensitive polymer, applied as a micro-pattern for μ-AJM process. The design and fabrication of the proposed micromixer is the first reported for such a device. Three glass layers were successfully bonded in a single step using a direct bonding method. These three bonded glass layers with micro-patterns etched on them were the realization of third-dimension feature on the micromixer design.

Microfabricated multilayer parylene‐C stencils for the generation of patterned dynamic co‐cultures

Co-culturing different cell types can be useful to engineer a more in vivo-like microenvironment for cells in culture. Recent approaches to generating cellular co-cultures have used microfabrication technologies to regulate the degree of cell-cell contact between different cell types. However, these approaches are often limited to the co-culture of only two cell types in static cultures. The dynamic aspect of cell-cell interaction, however, is a key regulator of many biological processes such as early development, stem cell differentiation, and tissue regeneration. In this study, we describe a micropatterning technique based on microfabricated multilayer parylene-C stencils and demonstrate the potential of parylene-C technology for co-patterning of proteins and cells with the ability to generate a series of at least five temporally controlled patterned co-cultures. We generated dynamic co-cultures of murine embryonic stem cells in culture with various secondary cell types that could be sequentially introduced and removed from the co-cultures. Our studies suggested that dynamic co-cultures generated by using parylene-C stencils may be applicable in studies investigating cellular interactions in controlled microenvironments such as studies of ES cell differentiation, wound healing and development.

Undercut structure fabricated by complementary-structure micropatterning technique for the passive-matrix display of organic light-emitting diodes

This paper reports a new patterning method, the complementary-structure micropatterning (CSMP) technique, to fabricate the undercut structures for the passive-matrix display of organic light-emitting diodes (OLEDs). First, the polyvinylpyrrolidone (PVP) stripe patterns with a trapeziform cross-section were formed by micromolding in capillaries. Then the photoresist was spin coated on the substrate with the patterned PVP stripes and developed in water. The PVP was dissolved and lifted off from the substrate with the photoresist deposited on it, resulting in the undercut structures of the remaining photoresist. The undercut structures with different configurations were obtained by adjusting the photoresist thickness. The undercut structures were further used as separators in the patterning of the passive-matrix display of OLEDs. No visible performance difference was observed compared with OLEDs patterned by the traditional method.

In situ micropatterning technique by cell crushing for co-cultures inside microfluidic biochips

To perform dynamic cell co-culture on micropatterned areas, we have developed a new type of "on chip and in situ" micropatterning technique. The microchip is composed of a 200 microm thick PDMS (polydimethylsiloxane) chamber at the top of which are located 100 mum thick microstamps. The PDMS chamber is bonded to a glass slide. After sterilization and cell adhesion processes, a controlled force is applied on the top of the PDMS chamber. Mechanically, the microstamps come into contact of the cells. Due to the applied force, the cells located under the microstamps are crushed. Then, a microfluidic perfusion is applied to rinse the microchip and remove the detached cells. To demonstrate the potential of this technique, it was applied successfully to mouse fibroblasts (Swiss 3T3) and liver hepatocarcinoma (HepG2/C3a) cell lines. Micropatterned areas were arrays of octagons of 150, 300 and 500 microm mean diameter. The force was applied during 30 to 60s depending on the cell types. After cell crushing, when perfusion was applied, the cells could successfully grow over the patterned areas. Cultures were successfully performed during 72 h of perfusion. In addition, monolayers of HepG2/C3a were micropatterned and then co cultured with mouse fibroblasts. Numerical simulations have demonstrated that the presence of the microstamps at the top of the PDMS chamber create non uniform flow and shear stress applied on the cells. Once fabricated, the main advantage of this technique is the possibility to use the same microchip several times for cell micropatterning and microfluidic co-cultures. This protocol avoids complex and numerous microfabrication steps that are usually required for micropatterning and microfluidic cell culture in the same time.

Reusable, reversibly sealable parylene membranes for cell and protein patterning

The patterned deposition of cells and biomolecules on surfaces is a potentially useful tool for in vitro diagnostics, high-throughput screening, and tissue engineering. Here, we describe an inexpensive and potentially widely applicable micropatterning technique that uses reversible sealing of microfabricated parylene-C stencils on surfaces to enable surface patterning. Using these stencils it is possible to generate micropatterns and copatterns of proteins and cells, including NIH-3T3 fibroblasts, hepatocytes and embryonic stem cells. After patterning, the stencils can be removed from the surface, plasma treated to remove adsorbed proteins, and reused. A variety of hydrophobic surfaces including PDMS, polystyrene and acrylated glass were patterned using this approach. Furthermore, we demonstrated the reusability and mechanical integrity of the parylene membrane for at least 10 consecutive patterning processes. These parylene-C stencils are potentially scalable commercially and easily accessible for many biological and biomedical applications.

Lithography Technique for Topographical Micropatterning of Collagen-Glycosaminoglycan Membranes for Tissue Engineering Applications

BACKGROUND: An adaptable technique for micropatterning biomaterial scaffolds has enormous implications in controlling cell function and in the development of tissue-engineered (TE) microvasculature. In this paper, we report a technique to embed microscale patterns onto a collagen-glycosaminoglycan (CG) membrane as a first step towards the creation of TE constructs with built-in microvasculature. METHOD OF APPROACH: The CG membranes were fabricated by homogenizing a solution of Type I bovine collagen and chondroitin 6-sulfate in acetic acid and vacuum filtering the solution subsequently. The micropatterning technique consisted of three steps: surface dissolution of base matrix using acetic acid solution, feature resolution by application of uniform pressure and feature stability by glutaraldehyde crosslinking. RESULTS: Application of the new technique yielded patterns in CG membranes with a spatial resolution in the order of 2-3 microns. We show that such a patterned matrix is conducive to the attachment of bovine aortic endothelial cells (BAEC's). CONCLUSIONS: The patterned membranes can be used for the development of complex three-dimensional TE products with built-in flow channels, as templates for topographically directed cell growth, or as a model system to study various microvascular disorders where feature scales are important. The new technique is versatile; topographical patterns can be custom-made for any predetermined design with high spatial resolution and the technique itself can be adapted for use with other scaffold materials.

Laser direct writing of thin-film copper structures as a modification of lithographic processes

This paper presents a flexible, mask-free and efficient technique for UV-laser micropatterning of photosensitive resist by laser direct writing (LDW). Photo resist spun on gold sputtered silicon wafers has been laser structured by a scanner guided 266nm DPSSL and electroplated. Ablation behaviour and optimum seed layer preparation in relation to parameters like pulse energy, scanning speed and number of scanned cycles and the electroplating results are discussed. The resulting adhesive strength was measured by a µ-sear device and the gold seed layer-plated copper interface investigated by SEM and EDX to explain correlation to identified bonding behaviour. Improved adhesive strength was observed with higher laser pulse energy and reduced number of cycle.

Nanocrystalline diamond lateral field emission diode fabrication by dual micropatterning technique

A dual-mask micropatterning process has been developed to fabricate nanocrystalline diamond lateral field emission diode with reduced reversed leakage current. In contrast to the all-diamond lateral device reported previously, this vacuum microelectronic device integrates a 6-finger lateral nanodiamond emitter cathode with a nickel metal anode on a SiO2 underlying substrate serving as the spacer material. In this configuration, the nitrogen-incorporated nanodiamond cathode has higher field enhancement factor, geometrically as well as materially, resulting in high rectification and excellent diode behavior. This lateral device processing further illustrates the viability of nanocrystalline diamond for device integration.

Comparative study and improvement of current cell micro-patterning techniques

The original micropatterning technique on gold, although very efficient, is not accessible to most biology labs and is not compatible with their techniques for image acquisition. Other solutions have been developed on silanized glass coverslips. These methods are still hardly accessible to biology labs and do not provide sufficient reproducibility to become incorporated in routine biological protocols. Here, we analyzed cell behavior on micro-patterns produced by various alternative techniques. Distinct cell types displayed different behavior on micropatterns, while some were easily constrained by the patterns others escaped or ripped off the patterned adhesion molecules. We report methods to overcome some of these limitations on glass coverslips and on plastic dishes which are compatible with our experimental biological applications. Finally, we present a new method based on UV crosslinking of adhesion proteins with benzophenone to easily and rapidly produce highly reproducible micropatterns without the use of a microfabricated elastomeric stamp.

Anisotropic three-dimensional peptide channels guide neurite outgrowth within a biodegradable hydrogel matrix

The objective of this study was to investigate the neurite guidance potential of concentration gradients of glycine–arginine–glycine–aspartic acid-serine (GRGDS) oligopeptides immobilized within three-dimensional patterned cylindrical volumes created in a biodegradable nerve guidance matrix. This was achieved using ultraviolet (UV) laser micropatterning of a hyaluronan (HA) hydrogel matrix modified with S-2-nitrobenzyl cysteine. Upon exposure to focused laser light, the 2-nitrobenzyl group was cleaved, exposing thiol groups which reacted with maleimide-terminated GRGDS exclusively within these laser-defined volumes. We show that the UV laser micropatterning technique can be used to create GRGDS peptide concentration gradients within the oligopeptide channels and that these channels guide neurite outgrowth from primary neural cells.

Micropatterning with aerosols: Application for biomaterials

Adhesion and proliferation behaviors of bovine aortic endothelial cells (BAECs) were investigated on surfaces micropatterned with peptides using a novel approach. This micropatterning technique allows modification of macroscopic three-dimensional (3D) biomaterials surfaces and exploits the semi-random properties of aerosols and the principles of liquid atomization. The possibility to control cell behaviors on polytetrafluoroethylene (PTFE) surfaces tailored with this micropatterning approach was evaluated. CGRGDS and CWQPPRARI peptides were selected for their adhesive, migration and spreading properties. Culture of BAECs on patterned PTFE showed the possibility of modulating cell behaviors. The study showed that CGRGDS spots with a diameter of 10+/-2 microm over a background of CWQPPRARI peptides was the most effective combination to enhance endothelialization of PTFE. This micropatterning technique is innovative, easily adaptable, simple, and rapid for covering large 3D areas.