Adipogenic differentiation of individual mesenchymal stem cell on different geometric micropatterns

Micropatterned surfaces are very useful to control cell microenvironment and investigate the physical effects on cell function. In this study, poly(vinyl alcohol) (PVA) micropatterns on polystyrene cell-culture plates were prepared using UV photolithography. Cell adhesive polystyrene geometries of triangle, square, pentagon, hexagon, and circle were surrounded by cell nonadhesive PVA to manipulate cell shapes. These different geometries had the same small surface areas for cell spreading. Human mesenchymal stem cells (MSCs) were cultured on the micropatterned surface, and the effect of cell geometry on adipogenic differentiation was investigated. MSCs adhered to the geometric micropatterns and formed arrays of single cell with different shapes. The distribution patterns of actin filaments were similar among these cell shapes and remolded during adipogenesis. The adipogenic differentiation potential of MSCs was similar on the small size triangular, square, pentagonal, hexagonal, and circular geometries according to lipid vacuoles staining. This simple micropatterning technique using photoreactive molecules will be useful for creating micropatterns of arbitrary design on an organic surface, and cell functions can be directly and systematically compared on a single surface without external factors resulting from separate cell culture and coating method.     

Micropatterning of functional conductive polymers with multiple surface chemistries in register

A versatile procedure is presented for fast and efficient micropatterning of multiple types of covalently bound surface chemistry in perfect register on and between conductive polymer microcircuits. The micropatterning principle is applied to several types of native and functionalized PEDOT (poly(3,4-ethylenedioxythiophene)) thin films. The method is based on contacting PEDOT-type thin films with a micropatterned agarose stamp containing an oxidant (aqueous hypochlorite) and applying a nonionic detergent. Where contacted, PEDOT not only loses its conductance but is entirely removed, thereby locally revealing the underlying substrate. Surface analysis showed that the substrate surface chemistry was fully exposed and not affected by the treatment. Click chemistry could thus be applied to selectively modify re-exposed alkyne and azide functional groups of functionalized polystyrene substrates. The versatility of the method is illustrated by micropatterning cell-binding RGD-functionalized PEDOT on low cell-binding PMOXA (poly(2-methyl-2-oxazoline)) to produce cell-capturing microelectrodes on a cell nonadhesive background in a few simple steps. The method should be applicable to a wide range of native and chemically functionalized conjugated polymer systems.     

Micropatterning of cell-repellent polymer on a glass substrate for the highly resolved virus microarray

The development of a simple and easily accessible method to control cellular behavior under a spatially controlled surface is critical for fundamental studies in biotechnology. We fabricated a microarray of Spodoptera frugiperda 9 (Sf9) cells on a glass surface by microcontact printing cell-repellent polymeric molecules of poly(ethylene glycol)-branched-poly(methyl methacrylate) as a template for cell micropatterning. The polymer micropatterns enabled the stable confinement of Sf9 cells on the surface, resulting in the formation of a cell microarray. Subsequently, the patterned Sf9 cells were infected with recombinant baculovirus modified with green fluorescent protein (GFP) to form a virus microarray, and GFP expression in the virus microarray was verified with confocal fluorescence microscopy.     

Novel high-resolution micropatterning for neuron culture using polylysine adsorption on a cell repellant, plasma-polymerized background

The ability to organize individual neurons and their processes in culture provides important benefits to both basic neuroscience research applications and the development of biomedical microdevices. While numerous methods have been used to produce such micropatterning of neurons and cells in general, there has yet been no method to simultaneously provide high-resolution patterns with high compliance of cells to desired patterns and good manufacturability. To develop such a process, this work used a plasma polymerized, nonfouling poly ethylene oxide (PEO)-like film to provide a cell repellant substrate on which cell adhesive micropatterns can be selectively laid down. While the use of plasma polymerized, organic films have been used for cell micropatterning, this process exploits the often-overlooked tendency for the surface of this PEO-like material to adsorb polylysine from aqueous solution while remaining nonfouling with respect to other species, such as bovine serum albumin (BSA) and immunoglobulin G (IgG). When the adsorption of polylysine was enhanced by brief plasma oxidation, which slightly alters the surface chemistry of the material, simple photolithographic liftoff could be used to micropattern stable, cell adhesive areas on an otherwise cell repellant background. We showed that the application of photolithography itself on the PEO-like material did not alter its chemical properties, nor did it result in the erosion of the micropatterned polylysine on its surface. Hippocampal neurons from embryonic mice flourished on these micropatterned substrates and exhibited viability comparable to neurons cultured on polylysine coated glass. Furthermore, the compliance of cell bodies and outgrowing neurites to the micropatterns was nearly perfect. In addition to providing cell adhesive regions, the micropatterned polylysine coating also served as a template mediating the immobilization of other bioactive species such as IgG and laminin. Using this "piggybacking" of laminin on polylysine, we were also able to culture and micropattern retinal ganglion cells (RGC).     

Microscale plasma-initiated patterning of electrospun polymer scaffolds

Microscale plasma-initiated patterning (μPIP) is a novel micropatterning technique used to create biomolecular micropatterns on polymer surfaces. The patterning method uses a polydimethylsiloxane (PDMS) stamp to selectively protect regions of an underlying substrate from oxygen plasma treatment resulting in hydrophobic and hydrophilic regions. Preferential adsorption of the biomolecules onto either the plasma-exposed (hydrophilic) or plasma-protected (hydrophobic) regions leads to the biomolecular micropatterns. In the current work, laminin-1 was applied to an electrospun polyamide nanofibrillar matrix following plasma treatment. Radial glial clones (neural precursors) selectively adhered to these patterned matrices following the contours of proteins on the surface. This work demonstrates that textured surfaces, such as nanofibrillar scaffolds, can be micropatterned to provide external chemical cues for cellular organization.     

"Smart" biopolymer for a reversible stimuli-responsive platform in cell-based biochips

The rapid response of a smart material surface to external stimuli is critical for application to cell-based biochips. The sharp and controllable phase transition of elastin-like polypeptide (ELP) enabled reversible cell adhesion on the surface by changing the temperature or salt concentration in the system. First, ELP micropatterns were prepared on a glass surface modified into aldehyde. The lysine-containing ELP (ELP-K) was genetically synthesized from E. coli for conjugation with the aldehyde on the glass surface. The phase transition of ELP was monitored in PBS and cell culture media using UV-visible spectroscopy, and a significant difference in transition temperature (Tt) was observed between the two solution systems. The micropatterning of ELP on the glass surface was performed by microcontact printing a removable polymeric template on the aldehyde-glass followed by incubation in ELP-K aqueous solution. The ELP micropatterns were imaged with atomic force microscopy and showed a monolayer thickness of approximately 4 nm. Imaging from time-of-flight secondary ion mass spectroscopy confirmed that the ELP molecules were successfully immobilized on the highly resolved micropatterns. Cell attachment and detachment could be reversibly controlled on the ELP surfaces by external stimuli. The hydrophobic phase above Tt resulted in the adhesion of fibroblasts, while the detachment of cells was induced by lowering the incubation temperature below Tt. The smart properties of ELP were reliable and reproducible, demonstrating potential applications in cell-based microdevices.     

Protein micropatterning on bifunctional organic-inorganic sol-gel hybrid materials

Active protein micropatterns and microarrays made by selective localization are popular candidates for medical diagnostics, such as biosensors, bioMEMS, and basic protein studies. In this paper, we present a simple fabrication process of thick (approximately 20 microm) protein micropatterning using capillary force lithography with bifunctional sol-gel hybrid materials. Because bifunctional sol-gel hybrid material can have both an amine function for linking with protein and a methacryl function for photocuring, proteins such as streptavidin can be immobilized directly on thick bifunctional sol-gel hybrid micropatterns. Another advantage of the bifunctional sol-gel hybrid materials is the high selective stability of the amine group on bifunctional sol-gel hybrid patterns. Because amine function is regularly contained in each siloxane oligomers, immobilizing sites for streptavidin are widely distributed on the surface of thick hybrid micropatterns. The micropatterning processes of active proteins using efficient bifunctional sol-gel hybrid materials will be useful for the development of future bioengineered systems because they can save several processing steps and reduce costs.     

Effect of Nb,Ti, and W ion implantation on surface properties and cell compatibility of silicon carbide

Silicon carbide is considered as a bio-inert semiconductor material; consequently, it has been proposed for potential applications in human body implantation. In this study, we study the effect of implanting different metal ions on the surface properties of silicon carbide single crystal. The valence states of the elements and the surface roughness of implanted SiC were studied using X-ray photoelectron spectroscopy and atomic force microscope, respectively. Osteoblastic MG-63 cells were utilized to characterize the cytocompatibility of ion implanted SiC. The results show that after Nb ion implantation on the SiC surface, it mainly exists in the form of Nb–C bond, Nb–O bond, and a small amount of metallic niobium. The titanium implanted on SiC primarily forms Ti-C bond and Ti-O bond. The tungsten implanted on SiC mostly presents as metallic tungsten and W–O bond. The roughness of silicon carbide single crystal is improved by ion implantation of all three metal ions. Ion implantation of titanium and niobium can improve the cell compatibility and hydrophilicity of silicon carbide, whereas ion implantation of tungsten reduces the cell compatibility and hydrophilicity of silicon carbide.     

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.     

On the Applicability of Plasma Assisted Chemical Micropatterning to Different Polymeric Biomaterials

A plasma process sequence has been developed to prepare chemical micropatterns on polymeric biomaterial surfaces. These patterns induce a guided localized cell layover at microscopic dimension. Two subsequent plasma steps are applied. In the first functionalization step a microwave ammonia plasma introduces amino groups to obtain areas for very good cell adhesion; the second passivation step combines pattern generation and creation of cell repelling areas. This downstream microwave hydrogen plasma process removes functional groups and changes the linkages of polymer chains at the outermost surfaces. Similar results have been obtained on different polymers including polystyrene (PS), polyhydroxyethylmethacrylate (PHEMA), polyetheretherketone (PEEK), polyethyleneterephthalate (PET) and polyethylenenaphthalate (PEN). Such a rather universal chemical structuring process could widen the availability of biomaterials with specific surface preparations.     

Ultra-rapid laser protein micropatterning: screening for directed polarization of single neurons

Protein micropatterning is a powerful tool for studying the effects of extracellular signals on cell development and regeneration. Laser micropatterning of proteins is the most flexible method for patterning many different geometries, protein densities, and concentration gradients. Despite these advantages, laser micropatterning remains prohibitively slow for most applications. Here, we take advantage of the rapid multi-photon induced photobleaching of fluorophores to generate sub-micron resolution patterns of full-length proteins on polymer monolayers, with sub-microsecond exposure times, i.e. one to five orders of magnitude faster than all previous laser micropatterning methods. We screened a range of different PEG monolayer coupling chemistries, chain-lengths and functional caps, and found that long-chain acrylated PEG monolayers are effective at resisting non-specific protein adhesion, while permitting efficient cross-linking of biotin-4-fluorescein to the PEG monolayers upon exposure to femtosecond laser pulses. We find evidence that the dominant photopatterning chemistry switches from a two-photon process to three- and four-photon absorption processes as the laser intensity increases, generating increasingly volatile excited triplet-state fluorophores, leading to faster patterning. Using this technology, we were able to generate over a hundred thousand protein patterns with varying geometries and protein densities to direct the polarization of hippocampal neurons with single-cell precision. We found that certain arrays of patterned triangles as small as neurite growth cones can direct polarization by impeding the elongation of reverse-projecting neurites, while permitting elongation of forward-projecting neurites. The ability to rapidly generate and screen such protein micropatterns can enable discovery of conditions necessary to create in vitro neural networks with single-neuron precision for basic discovery, drug screening, as well as for tissue scaffolding in therapeutics.     

A novel approach to the micropatterning of proteins using dewetting of polymer bilayers

The ability to control protein and cell positioning on a microscopic scale is crucial in many biomedical and bioengineering applications, such as tissue engineering and the development of biosensors. We propose here a novel, simple, and versatile method for the micropatterning of proteins. Micropatterned substrates are produced by the dewetting of a metastable polymer film on top of another polymer film. Selective adsorption, or micropatterning, of proteins can be achieved on such substrates by choosing pairs of polymers which differ in protein affinity. In this study, patterns were produced in bilayers of poly(methylmethacrylate) (PMMA) and polystyrene (PS), and of PMMA and octadecyltrichlorosilane (OTS). Fluorescence microscopy and atomic force microscopy (AFM) provide evidence that model proteins adsorb preferentially on isolated bio-adhesive (PS and OTS) micropatches in a protein-resistant (PMMA) matrix. "Inverse" protein patterns, containing non-adhesive (PMMA) islands in a protein-adhesive (PS) matrix can also be produced. Such micropatterned substrates could potentially be used in the development of biosensors and bioassays, and in the study of cell growth and motility.     

Biocompatible micropatterning of two different cell types

The spatial arrangement of individual cell types can now be routinely controlled using soft-lithography-based micropatterning of complementary cell-adhesive and cell-resistant patterns. However, the application of these tools in tissue engineering to recreate tissue complexity in vitro has been hampered by the challenge of finding noncytotoxic procedures for converting complementary cell-resistant regions that define the arrangement of the first cell type into cell-adhesive regions to allow for the attachment of other cell types. A polyelectrolyte assembly approach is presented here for the first time, which allows for this noncytotoxic conversion and, thus, micropatterning of two different cell types, for example, endothelial cells and fibroblasts, on biodegradable substrates. The flexibility of this approach is further demonstrated by inducing organized capillary formation by endothelial cells on micropatterned lines followed by subsequent assembly of fibroblasts.     

Micropatterns of double-layered nanofiber scaffolds with dual functions of cell patterning and metabolite detection

This paper describes the development of multi-functional nanofiber scaffolds consisting of multiple layers of nanofiber scaffolds and nanofiber-incorporated poly(ethylene glycol) (PEG) hydrogels. As a proof-of-concept demonstration, we fabricated micropatterned polymeric nanofiber scaffolds that were capable of simultaneously generating cellular micropatterns within a biomimetic environment and detecting cellular metabolic products within well-defined microdomains. To achieve this goal, we designed nanofiber scaffolds with both vertical and lateral microdomains. Vertically heterogeneous structures that were responsible for multi-functionality were realized by preparing double-layered nanofiber scaffolds consisting of an antibody-immobilized bottom layer of nanofibers and an upper layer of bare polystyrene (PS) nanofibers by a two-step sequential electrospinning process. Photopatterning of poly(ethylene glycol) (PEG) hydrogel on the electrospun nanofibers produced laterally heterogeneous micropatterned nanofiber scaffolds made of hydrogel microwells filled with a nanofibrous region, which is capable of generating cell and protein micropatterns due to the different interactions that cells and proteins have with PEG hydrogels and nanofibers. When HepG2 cells were seeded into resultant nanofiber scaffolds, cells selectively adhered within the 200 μm × 200 μm PS fiber microdomain and formed 180.2 ± 6.7 μm spheroids after 5 days of culture in the upper layer. Furthermore, immobilized anti-albumin in the bottom layer detected albumin secreted by micropatterned HepG2 cells with higher sensitivity than flat PS substrates, demonstrating successful accomplishment of dual functions using micropatterned double-layered nanofiber scaffolds.     

Polymeric optical microscreen for high-resolution surface plasmon resonance microarray imaging

In this study, we demonstrate a simple method to fabricate surface plasmon resonance (SPR) imaging microarrays using polymer micropatterns. The use of a micrometer-scale polymeric optical screen (microPOS) passivates the region deposited with polymer by completely removing SPR signals or by saturating the SPR signal far beyond the detection range of SPR imaging. Two schemes were suggested to create a surface microPOS by either micropatterning a thick insulating layer before deposition of a metal layer (complete removal of SPR) or after deposition of a metal layer (saturation of SPR signal). The two schemes were successfully applied for the imaging of biological adsorption with a high imaging resolution of approximately 100 microm/pattern and 10 microm separation. The validity of the system was verified with a biotin-streptavidin system as a model for the systematic binding of biomolecules. Further, binding of prostate-specific antigen (PSA) onto the anti-PSA SPR microarray was demonstrated as a useful method for detecting a cancer marker.     

Passive control of cell locomotion using micropatterns: the effect of micropattern geometry on the migratory behavior of adherent cells

Directed cell migration is critical to a variety of biological and physiological processes. Although simple topographical patterns such as parallel grooves and three-dimensional post arrays have been studied to guide cell migration, the effect of the dimensions and shape of micropatterns, which respectively represent the amount and gradient of physical spatial cues, on cell migration has not yet been fully explored. This motivates a quantitative characterization of cell migration in response to micropatterns having different widths and divergence angles. The changes in the migratory (and even locational) behavior of adherent cells, when the cells are exposed to physical spatial cues imposed by the micropatterns, are explored here using a microfabricated biological platform, nicknamed the "Rome platform". The Rome platform, made of a biocompatible, ultraviolet (UV) curable polymer (ORMOCOMP), consists of 3 μm thick micropatterns with different widths of 3 to 75 μm and different divergence angles of 0.5 to 5.0°. The migration paths through which NIH 3T3 fibroblasts move on the micropatterns are analyzed with a persistent random walk model, thus quantifying the effect of the divergence angle of micropatterns on cell migratory characteristics such as cell migration speed, directional persistence time, and random motility coefficient. The effect of the width of micropatterns on cell migratory characteristics is also extensively investigated. Cell migration direction is manipulated by creating the gradient of physical spatial cues (that is, divergence angle of micropatterns), while cell migration speed is controlled by modulating the amount of them (namely, width of micropatterns). In short, the amount and gradient of physical spatial cues imposed by changing the width and divergence angle of micropatterns make it possible to control the rate and direction of cell migration in a passive way. These results offer a potential for reducing the healing time of open wounds with a smart wound dressing engraved with micropatterns (or microscaffolds).     

Collagen adsorption and structure on polymer surfaces observed by atomic force microscopy

The structure and adsorption patterns of type I and type III collagen were imaged on various polymer substrates with atomic force microscopy. Type I collagen had higher adsorption on polystyrene than on a series of polymethacrylates and formed a network of tightly, interwoven strands. Upon adsorption to different polymethacrylates, with varying side chain lengths, the collagen molecules formed long, branching fibrils. Types I and III collagen had different adsorption patterns, in some cases, on the identical substrate material. For example, instead of forming a tightly packed network, type III forms long, branching fibers on the polystyrene surface. On other materials, such as poly(n-butyl methacrylate), the two types of collagen showed similar adsorption pattern and structure. Adsorbed collagen was also imaged on various blends of polystyrene and polymethacrylates to determine how the polymer surface chemical structure and surface topography mediates protein adsorption.     

Fabrication of interdigitated micropatterns of self-assembled polymer nanofilms containing cell-adhesive materials

Micropatterns of different biomaterials with micro- and nanoscale features and defined spatial arrangement on a single substrate are useful tools for studying cellular-level interactions, and recent reports have highlighted the strong influence of scaffold compliance in determining cell behavior. In this paper, a simple yet versatile and precise patterning technique for the fabrication of interdigitated micropatterns of nanocomposite multilayer coatings on a single substrate is demonstrated through a combination of lithography and layer-by-layer (LbL) assembly processes, termed polymer surface micromachining (PSM). The first nanofilm pattern is constructed using lithography, followed by LbL multilayer assembly and lift-off, and the process is repeated with optical alignment to obtain interdigitated patterns on the same substrate. Thus, the method is analogous to surface micromachining, except that the deposition materials are polymers and biological materials that are used to produce multilayer nanocomposite structures. A key feature of the multilayers is the capability to tune properties such as stiffness by appropriate selection of materials, deposition conditions, and postdeposition treatments. Two- and four-component systems on glass coverslips are presented to demonstrate the versatility of the approach to construct precisely defined, homogeneous nanofilm patterns. In addition, an example of a complex system used as a testbed for in vitro cell adhesion and growth is provided: micropatterns of poly(sodium 4-styrenesulfonate)/poly-L-lysine hydrobromide (PSS/PLL) and secreted phospholipase A(2)/poly(ethyleneimine) (sPLA(2)/PEI) multilayers. The interdigitated square nanofilm array patterns were obtained on a single coverslip with poly(diallyldimethylammonium chloride) (PDDA) as a cell-repellent background. Cell culture experiments show that cortical neurons respond and bind specifically to the sPLA(2) micropatterns in competition with PLL micropatterns. The fabrication and the initial biological results on the nanofilm micropatterns support the usefulness of this technique for use in studies aimed at elucidating important biological structure-function relationships, but the applicability of the fabrication method is much broader and may impact electronics, photonics, and chemical microsystems.     

Area-selective microscale metallization on porous anodic oxide film of aluminium

A new method for micropatterning of metallic patterns on porous anodic oxide film of aluminium is described. The porous anodic oxide film was impregnated with organic dye and palladium ions before the hydrothermal pore-sealing. The surface layers formed during the pore-sealing, i.e. outer acicular hydroxide layer and a compact intermediate sub-layer trap the palladium ion underneath the layers. Exposing the palladium enriched area by the help of laser beam followed by electroless nickel deposition results the deposition of nickel on the laser-exposed part. Thickness of the deposits can be up to about 2–3 μm, after about the 20 min of immersion in electroless nickel plating bath. The metallic micropatterns, formed by the method are crack free, smooth and uniform over extended length.     

Micropatterning of polystyrene nanoparticles and its bioapplications

Micropatterning of biomolecules forms the basis of cell culture, biosensor and microarray technology. Currently, the most widely used techniques are photoresist lithography, soft lithography or using robots which all involve multi-step surface modification directly on a planar substrate. Here we report a method to pattern biomolecules through self-assembling polystyrene nanoparticles in arrayed microwells on a solid surface to form well-ordered patterning, followed by attaching biomolecules to the assembled nanoparticles. The formation of colloidal patterns depends on capillary force, surface wettability and physical confinement. This method can be used for micropatterning a variety of biomolecules such as protein and antibody.