Rehydration of Polymeric,Aqueous, Biphasic System Facilitates High Throughput Cell Exclusion Patterning for Cell Migration Studies

This paper describes a cell‐exclusion patterning method facilitated by a polymeric aqueous two‐phase system. The immersion aqueous phase (polyethylene glycol) containing cells rehydrates a dried disk of the denser phase (dextran) on the substrate to form a dextran droplet. With the right properties of the phase‐forming polymers, the rehydrating droplet remains immiscible with the immersion phase. Proper formulation of the two‐phase system ensures that the interfacial tension between the rehydrating droplet and the surrounding aqueous phase prevents cells from crossing the interface so that cells only adhere to the regions of the substrate around the dextran phase droplet. Washing out the patterning two‐phase reagents reveals a cell monolayer containing a well‐defined circular gap that serves as the migration niche for cells of the monolayer. Migration of cells into the cell‐excluded area is readily visualized and quantified over time. A 96‐well plate format of this “gap healing” migration assay demonstrates the ability to detect inhibition of cell migration by known cytoskeleton targeting agents. This straightforward method, which only requires a conventional liquid handler and readily prepared polymer solutions, opens new opportunities for high throughput cell migration assays.     

Imaging Surface Immobilization Chemistry: Correlation with Cell Patterning on Non‐Adhesive Hydrogel Thin Films

High‐fidelity surface functional group (e.g., N‐hydroxysuccinimide (NHS) reactive ester) patterning is readily and reliably achieved on commercial poly(ethylene glycol) (PEG)‐based polymer films already known to exhibit high performance non‐fouling properties in full serum and in cell culture conditions. NHS coupling chemistry co‐patterned with methoxy‐capped PEG using photolithographic methods is directly spatially imaged using imaging time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) and principal components statistical analysis. Patterned NHS surface reactive zones are clearly resolved at high sensitivity despite the complexity of the polymer matrix chemistry. ToF‐SIMS imaging also reveals the presence of photo‐resist residue remaining from typical photolithography processing methods. High cross‐correlation between various ion‐derived ToF‐SIMS images is observed, providing sensitive chemical corroboration of pattern chemistry and biological reactivity in complex milieu. Surface‐specific protein coupling is observed first by site‐selective reaction of streptavidin with NHS patterns, followed by identical patterns of biotinylated Alexa‐labeled albumin coupling. This suggests that streptavidin immobilized on the patterns remains bioactive. Fluorescently labeled full serum is shown to react selectively with NHS‐reactive regions, with minimal signal from methoxy‐capped regions. Insufficient serum is adsorbed under any conditions to these surfaces to support cell attachment in serum‐containing media. This reflects the high intrinsic non‐adsorptive nature of this chemistry. Fibroblasts attach and proliferate in serum culture only when a cell adhesion peptide (RGD) is first grafted to NHS regions on the PEG‐based surfaces. Longer‐term serum‐based cell culture retains high cell‐pattern fidelity that correlates with chemical imaging of both the NHS and RGD patterns and also lack of cell adhesion to methoxy‐capped regions. Cell staining shows orientation of adherent cells within the narrow patterned areas. Cell patterns are consistently retained beyond 15 days in serum media.     

Cell Adhesion and Cellular Patterning on a Self‐Assembled Monolayer of Zeolite L Crystals

Chemically functionalized self‐assembled monolayers made by disk‐shaped zeolite L nanocrystals are used as models for biocompatible surfaces to study cell‐adhesion behavior. Different chemical groups lead to different cellular behavior and fluorescent‐molecule‐loaded zeolites allow the position of the cells to be determined. Furthermore, a patterned monolayer of asymmetrically functionalized zeolite L obtained by microcontact chemistry is used to grow cells. A spatial recognition of the cells, which proliferate only on the bioactive‐molecule‐functionalized stripes, is possible.     

High‐Resolution Patterning of Hydrogels in Three Dimensions using Direct‐Write Photofabrication for Cell Guidance

The development of three‐dimensional, spatially defined neuronal cultures that mimic chemical and physical attributes of native tissue is of considerable interest for various applications, including the development of tailored neuronal networks and clinical repair of damaged nerves. Here, the use of multiphoton excitation to photocrosslink protein microstructures within three‐dimensional, optically transparent hydrogel materials, such as those based on hyaluronic acid, is reported. Multiphoton excitation confines photocrosslinking to a three‐dimensional voxel with submicron spatial resolution, enabling fabrication of protein matrices with low‐ to sub‐micrometer feature sizes by scanning the focus of a laser relative to the reagent solution. These methods can be used to create complex three‐dimensional architectures that provide both chemical and topographical cues for cell culture and guidance, providing for the first time a means to direct cell adhesion and migration on size scales relevant to in vivo environments. Using this approach, guidance of both dorsal root ganglion cells (DRGs) and hippocampal neural progenitor cells (NPCs) along arbitrary, three‐dimensional paths is demonstrated.     

User‐Friendly Universal and Durable Subcellular‐Scaled Template for Protein Binding: Application to Single‐Cell Patterning

A new method for subcellular‐sized protein patterning on a SiOx substrate is demonstrated by dip‐pen nanolithography printed aldehyde‐terminated alkylsilane template. The aldehyde‐silane template is stable and durable; for example, subcellular scaled IgG protein array can be obtained using one‐year old aldehyde‐silane template. Moreover, single cell patterning is successfully carried out by extracellular material (ECM) protein microarray and nanoarray fabricated on an aldehyde‐silane template. With more than half of chance, single‐ or double‐cells are successfully attached on fibronectin protein nanoarrays in 21 × 21 μm ² (7 × 7 dot array) and 42 × 42 μm² (14 × 14 dot array). The fibronectin nanoarray with small area (21 × 21 μm²) shows the more rate of single cell attachment. Therefore, it is also demonstrated that cell patterning can be controlled by adjusting the nanostructure of ECM materials.     

Controllable Patterning of Different Cells Via Optical Assembly of 1D Periodic Cell Structures

Flexible patterning of different cells into designated locations with direct cell–cell contact at single‐cell patterning precision and control is of great importance, however challenging, for cell patterning. Here, an optical assembly method for patterning of different types of cells via direct cell–cell contact at single‐cell patterning precision and control is demonstrated. Using Escherichia coli and Chlorella cells as examples, different cells are flexibly patterned into 1D periodic cell structures (PCSs) with controllable configurations and lengths, by periodically connecting one type of cells with another by optical force. The patterned PCSs can be flexibly moved and show good light propagation ability. The propagating light signals can be detected in real‐time, providing new opportunities for the detection of transduction signals among patterned cells. This patterning method is also applicable for cells of other kinds, including mammalian/human cells.     

Direct UV Patterning of Electronically Active Fullerene Films

We utilize UV light for the attainment of high‐resolution, electronically active patterns in [6,6]‐phenyl C₆₁‐butyric acid methyl ester (PCBM) films. The patterns are created by directly exposing selected parts of a solution‐cast PCBM film to UV light, and thereafter developing the film by immersing it in a tuned developer solution. We demonstrate that it is possible to attain complex, large‐area PCBM structures with a smallest demonstrated‐feature size of 1 μm by this method, and that the patterned PCBM material exhibits a high average electron mobility (1.2 × 10^?2 cm² V^?1 s^?1) in transistor experiments. The employment of UV light for direct patterning of PCBM for electronic applications is attractive, because PCBM exhibits high absorption in the UV range, and no sacrificial photoresist is needed. The patterning is achieved through the transformation by UV light of the soluble PCBM monomers into insoluble dimers with retained attractive electronic properties.     

UV‐Triggered Polymerization,Deposition, and Patterning of Plant Phenolic Compounds

Plant‐derived phenolic compounds, rich in catechol and pyrogallol moieties, can form multifunctional coatings on various substrates following polymerization under mildly alkaline conditions. Despite many appealing features of such coatings, the difficulty to control polymerization of phenolic compounds spatially and temporally limits their number of potential applications. In this study, it is demonstrated that UV irradiation can trigger oxidative polymerization and deposition of plant‐derived phenolic compounds, which opens the possibility to create 2D gradients and patterns of polyphenol coatings and control this polymerization temporally. UV–vis spectroscopy, electrospray ionization mass spectrometry, and cyclic voltammetry analyses are used to investigate the UV‐induced polymerization of several plant‐derived phenolic compounds including pyrogallol, tannic acid, caffeic acid, and gallic acid. Formation of polyphenol coatings on polar and nonpolar substrates after UV irradiation has been studied using water contact angle measurements, atomic force microscopy, time of flight secondary ion mass spectrometry, and X‐ray photoelectron spectroscopy (XPS). The possibility to use UV‐light to accelerate polymerization of phenolic compounds and perform micropatterning can extend the scope of potential applications of the large class of structurally diverse plant‐derived phenolic compounds.     

Bio‐Orthogonally Crosslinked,Engineered Protein Hydrogels with Tunable Mechanics and Biochemistry for Cell Encapsulation

Covalently‐crosslinked hydrogels are commonly used as 3D matrices for cell culture and transplantation. However, the crosslinking chemistries used to prepare these gels generally cross‐react with functional groups present on the cell surface, potentially leading to cytotoxicity and other undesired effects. Bio‐orthogonal chemistries have been developed that do not react with biologically relevant functional groups, thereby preventing these undesirable side reactions. However, previously developed biomaterials using these chemistries still possess less than ideal properties for cell encapsulation, such as slow gelation kinetics and limited tuning of matrix mechanics and biochemistry. Here, engineered elastin‐like proteins (ELPs) are developed that crosslink via strain‐promoted azide‐alkyne cycloaddition (SPAAC) or Staudinger ligation. The SPAAC‐crosslinked materials form gels within seconds and complete gelation within minutes. These hydrogels support the encapsulation and phenotypic maintenance of human mesenchymal stem cells, human umbilical vein endothelial cells, and murine neural progenitor cells. SPAAC‐ELP gels exhibit independent tuning of stiffness and cell adhesion, with significantly improved cell viability and spreading observed in materials containing a fibronectin‐derived arginine‐glycine‐aspartic acid (RGD) domain. The crosslinking chemistry used permits further material functionalization, even in the presence of cells and serum. These hydrogels are anticipated to be useful in a wide range of applications, including therapeutic cell delivery and bioprinting.     

Biomolecule Arrays Using Functional Combinatorial Particle Patterning on Microchips

Biofunctionalization of surfaces in a microarray format has revolutionized biological assay applications. Here, a microarray system based on a microelectronic chip is presented that allows for a versatile combinatorial in situ molecule synthesis with very high density. Successfully demonstrating an application for peptide array synthesis, the method offers a compact approach, high combinatorial freedom, and, due to the intrinsic alignment, high and reproducible precision. Patterning the chip surface with different microparticle types which imbed different monomers, several thousand different molecule types can be simultaneously elongated layer‐by‐layer by coupling the particle imbedded monomers to the molecules growing on the chip surface. This technique has the potential for a wide application in combinatorial chemistry, as long as the desired monomeric building blocks are compatible with the chemical process.     

Optimal Radius of Exclusion Zone for Dense Cognitive Small Cell Networks

Network densification via small cell deployments can enhance the network capacity in a cost-effective manner. Co-tier and cross-tier interferences form barriers to boost the network performance. In this paper, an interference coordination scheme with the aid of cognitive radio is proposed for dense small cell networks. Based on the theory of stochastic geometry, the network spectral efficiency is derived as a function of the radius of exclusion zone. Furthermore, the optimal radius of exclusion zone that maximizes system spectral efficiency is obtained by one-dimension searching. Simulation results show that the proposed interference coordination scheme with the optimal exclusion zone outperforms the existing ones in terms of system spectral efficiency.     

Plastic Electronic Devices Through Line Patterning of Conducting Polymers

A novel method for the preparation of transparent conducting‐polymer patterns on flexible substrates is presented. This method, line patterning, employs mostly standard office equipment, such as drawing software, a laser printer, and commercial overhead transparencies, together with a solution or dispersion of a conducting polymer. The preparation of a seven‐segment polymer‐dispersed liquid‐crystal display using electrodes of the conducting polymer poly(3,4‐ethylenedioxythiophene) doped with poly(4‐styrene sulfonate) (PEDOT/PSS) is described in detail. Furthermore, a method to fabricate an eleven‐key push‐button array for keypad applications is presented. Properties of the electrode films and patterns are discussed using microscopy images, atomic force microscopy, conductivity measurements, and tests of film stability.