Examining the lateral displacement of HL60 cells rolling on asymmetric P-selectin patterns

The lateral displacement of cells orthogonal to a flow stream by rolling on asymmetrical receptor patterns presents a new opportunity for the label-free separation and analysis of cells. Understanding the nature of cell rolling trajectories on such substrates is necessary to the engineering of substrates and the design of devices for cell separation and analysis. Here, we investigate the statistical nature of cell rolling and the effect of pattern geometry and flow shear stress on cell rolling trajectories using micrometer-scale patterns of biomolecular receptors with well-defined edges. Leukemic myeloid HL60 cells expressing the PSGL-1 ligand were allowed to flow across a field of patterned lines fabricated using microcontact printing and functionalized with the P-selectin receptor, leveraging both the specific adhesion of this ligand-receptor pair and the asymmetry of the receptor pattern inclination angle with respect to the fluid shear flow direction (α = 5, 10, 15, and 20°). The effects of the fluid shear stress magnitude (τ = 0.5, 1, 1.5, and 2.0 dyn/cm(2)), α, and P-selectin incubation concentration were quantified in terms of the rolling velocity and edge tracking length. Rolling cells tracked along the inclined edges of the patterned lines before detaching and reattaching on another line. The detachment of rolling cells after tracking along the edge was consistent with a Poisson process of history-independent interactions. Increasing the edge inclination angle decreased the edge tracking length in an exponential manner, contrary to the shear stress magnitude and P-selectin incubation concentration, which did not have a significant effect. On the basis of these experimental data, we constructed an empirical model that predicted the occurrence of the maximum lateral displacement at an edge angle of 7.5°. We also used these findings to construct a Monte Carlo simulation for the prediction of rolling trajectories of HL60 cells on P-selectin-patterned substrates with a specified edge inclination angle. The prediction of lateral displacement in the range of 200 μm within a 1 cm separation length supports the feasibility of label-free cell separation via asymmetric receptor patterns in microfluidic devices.     

Probing the Adhesion of Hepatocellular Carcinoma HepG2 and SK‐Hep‐1 Cells

Carcinoma cell differentiation stage is an important indicator of cell behavior. For example, cell mobility is much higher for poorly‐differentiated hepatocellular carcinoma SK‐Hep‐1 cells than for well‐differentiated HepG2 cells. In this study, we have cultured HepG2 and SK‐Hep‐1 cells on chemically patterned polydimethylsiloxane (PDMS) substrates to observe differences in the adhesion properties and cell structure that occur due to the patterns. Both cell lines showed a preference for the hydrophobic regions on the patterned PDMS surface with SK‐Hep‐1 cells achieving a higher density than HepG2 for the same cell‐count solutions. Further, SK‐Hep‐1 cells adopted the square or hexagonal shape of the surface patterns while HepG2 cells maintained their more rounded shape. AFM force measurement arrays were also performed on the cell surfaces to measure and map adhesion values between the tip and cell surface membrane. These results demonstrate that, in addition to cell shape and size, adhesion expression in hepatocellular carcinoma cells is differentiation stage dependent. Further, the ability of the SK‐Hep‐1 cells to adopt the shape of the substrate pattern indicates they are more structurally labile, which may contribute to their higher mobility.     

Patterned surfaces segregate compliant microcapsules

For both biological cells and synthetic microcapsules, mechanical stiffness is a key parameter since it can reveal the presence of disease in the former case and the quality of the fabricated product in the latter case. To date, however, assessing the mechanical properties of such micron-scale particles in an efficient, cost-effective means remains a critical challenge. By developing a three-dimensional computational model of fluid-filled, elastic spheres rolling on substrates patterned with diagonal stripes, we demonstrate a useful method for separating cells or microcapsules by their compliance. In particular, we examine the fluid-driven motion of these capsules over a hard adhesive surface that contains soft stripes or a weakly adhesive surface that contains "sticky" stripes. As a result of their inherently different interactions with the heterogeneous substrate, particles with dissimilar stiffness are dispersed to distinct lateral locations on the surface. Since mechanically and chemically patterned surfaces can be readily fabricated through soft lithography and can easily be incorporated into microfluidic devices, our results point to a facile method for carrying out continuous "on the fly" separation processes.     

Cellular responses to patterned poly(acrylic acid) brushes

We use patterned poly(acrylic acid) (PAA) polymer brushes to explore the effects of surface chemistry and topography on cell-surface interactions. Most past studies of surface topography effects on cell adhesion have focused on patterned feature sizes that are larger than the dimensions of a cell, and PAA brushes have been characterized as cell repellent. Here we report cell adhesion studies for RBL mast cells incubated on PAA brush surfaces patterned with a variety of different feature sizes. We find that when patterned at subcellular dimensions on silicon surfaces, PAA brushes that are 30 or 15 nm thick facilitate cell adhesion. This appears to be mediated by fibronectin, which is secreted by the cells, adsorbing to the brushes and then engaging cell-surface integrins. The result is detectable accumulation of plasma membrane within the brushes, and this involves cytoskeletal remodeling at the cell-surface interface. By decreasing brush thickness, we find that PAA can be 'tuned' to promote cell adhesion with down-modulated membrane accumulation. We exemplify the utility of patterned PAA brush arrays for spatially controlling the activation of cells by modifying brushes with ligands that specifically engage IgE bound to high-affinity receptors on mast cells.     

The deformation and adhesion of randomly rough and patterned surfaces

Using a surface forces apparatus (SFA) and an atomic force microscope (AFM) we have studied the effects of surface roughness (root-mean-square (RMS) roughness between 0.3 and 220 nm) on the "contact mechanics", which describes the deformations and loading and unloading adhesion forces, of various polymeric surfaces. For randomly rough, moderately stiff, elastomeric surfaces, the force-distance curves on approach and separation are nearly reversible and almost perfectly exponentially repulsive, with an adhesion on separation that decreases only slightly with increasing RMS. Additionally, the magnitude of the preload force is seen to play a large role in determining the measured adhesion. The exponential repulsion likely arises from the local compressions (fine-grained nano- or submicron-scale deformations) of the surface asperities. The resulting characteristic decay lengths of the repulsion scale with the RMS roughness and correlate very well with a simple finite element method (FEM) analysis based on actual AFM topographical images of the surfaces. For "patterned" surfaces, with a nonrandom terraced structure, no similar exponential repulsion is observed, suggesting that asperity height variability or random roughness is required for the exponential behavior. However, the adhesion force or energy between two "patterned" surfaces fell off dramatically and roughly exponentially as the RMS increased, likely owing to a significant decrease in the contact area which in turn determines their adhesion. For both types of rough surfaces, random and patterned, the coarse-grained (global, meso- or macroscopic) deformations of the initially curved surfaces appear to be Hertzian.     

Electroactive self-assembled monolayers that permit orthogonal control over the adhesion of cells to patterned substrates

This article describes an electroactive substrate that displays two independent dynamic functions for controlling the adhesion of cells. The approach is based on self-assembled monolayers on gold that are patterned into regions presenting the Arg-Gly-Asp peptide cell adhesion ligand. The patterned regions differ in the electrochemical properties of the linkers that tether the peptides to the monolayer. In this work, three distinct chemistries are employed that provide for release of the ligand on application of a negative potential, release of the ligand on application of a positive potential, and no change in response to a potential. Cells were allowed to attach to a monolayer patterned into circular regions comprising the three chemistries. Treatment with electric potentials of 650 or -650 mV resulted in the selective release of adherent cells only from regions that display the relevant electroactive groups. This example establishes the preparation of dynamic substrates with multiple functions and will be important to preparing model cultures derived from multiple cell types, with control over the temporal interactions of each cell population.     

In situ photopolymerization of biomaterials by thiol-yne click chemistry

The thiol-yne click chemistry reaction has been used for the in situ photocrosslinking of an aliphatic hyperbranched polyester. The biocompatibility of the resulting networks has been studied and marked cytotoxicity was not found for HeLa (human cervical carcinoma) tumoral cells and COS7 fibroblasts. The photoinduced thiol-yne process allows the generation of patterned structures with different geometries in films by DLW and these materials can be used as substrates for cell adhesion. The influence of the substrate geometry on cell adhesion has been studied by culturing cells onto these substrates and a preference for the photopatterned polymeric material can be seen in some of the structures by contrast phase microscopy. Actin and vinculin fluorescent staining revealed different adhesion behavior for HeLa cells and COS7 fibroblasts and this could be assigned to the different motility of cells. The thiol-yne photoreaction has proven to be an attractive approach for the preparation of micropatterned biomaterials.     

Dual silane surface functionalization for the selective attachment of human neuronal cells to porous silicon

Porous silicon (pSi) surfaces were chemically micropatterned through a combination of photolithography and surface silanization reactions. This patterning technique produces discretely defined regions on a pSi surface functionalized with a specific chemical functionality, and the surrounding surface displays a completely different functionality. The generated chemical patterns were characterized by a combination of IR microscopy and the conjugation of two different fluorescent organic dyes. Finally, the chemically patterned pSi surface was used to direct the attachment of neuronal cells to the surface. This patterning strategy will be useful for the development of high-throughput platforms for investigating cell behavior.     

Short peptides enhance single cell adhesion and viability on microarrays

Single cell patterning holds important implications for biology, biochemistry, biotechnology, medicine, and bioinformatics. The challenge for single cell patterning is to produce small islands hosting only single cells and retaining their viability for a prolonged period of time. This study demonstrated a surface engineering approach that uses a covalently bound short peptide as a mediator to pattern cells with improved single cell adhesion and prolonged cellular viability on gold patterned SiO2 substrates. The underlying hypothesis is that cell adhesion is regulated by the type, availability, and stability of effective cell adhesion peptides, and thus covalently bound short peptides would promote cell spreading and, thus, single cell adhesion and viability. The effectiveness of this approach and the underlying mechanism for the increased probability of single cell adhesion and prolonged cell viability by short peptides were studied by comparing cellular behavior of human umbilical cord vein endothelial cells on three model surfaces whose gold electrodes were immobilized with fibronectin, physically adsorbed Arg-Glu-Asp-Val-Tyr, and covalently bound Lys-Arg-Glu-Asp-Val-Tyr, respectively. The surface chemistry and binding properties were characterized by reflectance Fourier transform infrared spectroscopy. Both short peptides were superior to fibronectin in producing adhesion of only single cells, whereas the covalently bound peptide also reduced apoptosis and necrosis of adhered cells. Controlling cell spreading by peptide binding domains to regulate apoptosis and viability represents a fundamental mechanism in cell-materials interaction and provides an effective strategy in engineering arrays of single cells.     

Local control of protein binding and cell adhesion by patterned organic thin films

Control of the cell adhesion and growth on chemically patterned surfaces is important in an increasing number of applications in biotechnology and medicine, for example implants, in-vitro cellular assays, and biochips. This review covers patterning techniques for organic thin films suitable for site-directed guidance of cell adhesion to surfaces. Available surface patterning techniques are critically evaluated, with special emphasis on surface chemistry that can be switched in time and space during cultivation of cells. Examples from the authors’ laboratory include the use of cell-repellent self-assembled monolayers (SAM) terminated by oligoethylene glycol (OEG) units and the lifting of the cell repellent properties by use of electrogenerated Br₂/HOBr which can be performed with positionable microelectrodes. Structural changes of the SAM were analyzed by polarization-modulated infrared reflection absorption spectroscopy (PM IRRAS). Use of a soft array system of individually addressable microelectrodes enables formation of flexible and complex patterns in a short time and has the potential for further acceleration of probe-induced local manipulation of cell adhesion.     

Wetting films on chemically patterned surfaces

The behavior of thin wetting films on chemically patterned surfaces was investigated. The patterning was performed by means of imprinting of micro-grid on methylated glass surface with UV-light (λ=184.8 nm). Thus imprinted image of the grid contained hydrophilic cells and hydrophobic bars on the glass surface. For this aim three different patterns of grids were utilized with small, medium and large size of cells. The experiment showed that the drainage of the wetting aqueous films was not affected by the type of surface patterning. However, after film rupturing in the cases of small and medium cells of the patterned grid the liquid from the wetting film underwent fast self-organization in form of regularly ordered droplets covering completely the cells of the grid. The droplets reduced significantly their size upon time due to evaporation. In the cases of the largest cell grid, a wet spot on the place of the imprinted grid was formed after film rupturing. This wet spot disassembled slowly in time. In addition, formation of a periodical zigzag three-phase contact line (TPCL) was observed. This is a first study from the planned series of studies on this topic.     

Engineering Photocleavable Protein-decorated Hydrogels to Regulate Cell Adhesion and Migration

Cell adhesion and migration play essential roles in tissue development and maintenance, and abnormal cell migration is involved in life-threatening diseases, including vascular disease, tumor formation, and metastasis. The advances in hydrogel-based 3D cell culture development facilitated the investigation of cell motility behavior, including cell-cell and cell-matrix adhesion and cell migration in a microenvironment more related to in vivo situations. Establishing advanced methods for these in vitro studies is thus necessary. Photo-sensitive proteins show advantages in remote and non-invasive regulation of hydrogels' properties, and thus are of great potential in regulating 3D cultured cells' behavior. In the presented study, we engineered photocleavable protein(PhoCl)-decorated hydrogels to regulate cell adhesion and migration of MDA-MB-231. The integrin-binding motif RGD was fused to the PhoCl and was decorated on the hydrogel. After being exposed to light at 405 nm, the PhoCl was cleaved and the RGD motif was released, resulting in detachment of the binding cells. The regulatory effect of the light illumination showed a time-dependent and cell density-dependent manner. Furthermore, the elimination of RGD by patterned light exposure completely suspended the cell migration to the corresponding region, suggesting a controllable regulation of the cell migration direction.     

Controlling cell attachment selectively onto biological polymer-colloid templates using polymer-on-polymer stamping

A new patterning approach using polymer-on-polymer stamping (POPS) has been developed to fabricate polymer-colloid templates for controlling selective cell attachment. In this paper, a polyamine surface patterned onto a poly(acrylic acid)/poly(allylamine hydrochloride) (PAA/PAH) cell resistant multilayer platform serves as a template for the deposition of close- or loose-packed colloidal particles. Peptides containing the RGD adhesion sequence were used to modify the PAH/colloid surface for specific cell attachment. Cell behavior was studied by varying colloidal packing array density, pattern geometry, and surface chemistry. It was found that loose-packed RGD-modified colloidal arrays enhance cell adhesion, as observed through the development of focal adhesion contacts and orientation of actin stress fibers, but close-packed colloidal arrays induce a rounded and nonadhesive cell morphology and yield a smaller number of attached cells. On loose-packed arrays, cells adjust their shapes to the pattern geometry when the stripe width is smaller than 50 microm and increase their extent of attachment when the concentration of surface RGD peptides is increased. This new biomaterials system allows the examination of cell behavior as a function of RGD surface distribution on the molecular to micrometer scale and reveals cellular response to different surface roughnesses.     

Micro- and nanometer-scale patterned surface in a microchannel for cell culture in microfluidic devices

A novel microdevice which had a micro- and nanometer-scale patterned surface for cell adhesion in a microchip was developed. The surface had a metal pattern fabricated by electron-beam lithography and metal sputtering and a chemical pattern consisting of a self-assembled monolayer of alkanethiol. The metal patterned surface had a gold stripe pattern which was as small as 300 nm wide and 150 nm high and both topography and chemical properties could be controlled. Mouse fibroblast NIH/3T3 cells were cultured on the patterned surface and elongated along the gold stripes. These cells recognized the size of the pattern and the chemical properties on the pattern though it was much smaller than they were. There was satisfactory cell growth under fresh medium flow in the microchip. The combination of the patterned surface and the microchip provides cells with a novel environment for their growth and will facilitate many cellular experiments. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nanometer-scale patterned surfaces for control of cell adhesion.

A novel cell-adhesion surface, controlled by nanometer-scale topography and chemical patterning, was developed using semiconductor fabrication methods and the formation of self-assembled monolayers. The patterned surface had a sharp contrast between the adsorption and non-adsorption of proteins and cells, and the contrast could be maintained for more than 10 days. The patterning method could easily realize a single cell array and control of the cell morphology. The nanometer-scale patterned surface could control cell adhesion and proliferation. Using the patterned surface will contribute to studies about cell-surface interactions.     

Real-time characterization of cytotoxicity using single-cell impedance monitoring

Cellular impedance sensors have attracted great attention as a powerful characterization tool for real-time, label-free detection of cytotoxic agents. However, impedance measurements with conventional cell-based sensors that host multiple cells on a single electrode neither provide optimal cell signal sensitivity nor are capable of recording individual cell responses. Here we use a single-cell based platform to monitor cellular impedance on planar microelectrodes to characterize cellular death. In this study, individual cells were selectively patterned on microelectrodes with each hosting one live cell through ligand-mediated natural cell adhesion. Changes in cellular morphology and cell-electrode adherence were monitored after the patterned cells were treated with varying concentrations of hydrogen peroxide, sodium arsenite, and disodium hydrogen arsenate, three potent toxicants related to neurotoxicity and oxidative stress. At low toxicant concentrations, impedance waveforms acquired from individual cells showed variable responses. A time- and concentration-dependent response was seen in the averaged single-cell impedance waveform for all three toxicants. The apoptosis and necrosis characterizations were performed to validate cell impedance results. Furthermore, time constants of apoptosis and necrosis in response to toxicant exposure were analytically established using an equivalent circuit model that characterized the mechanisms of cell death.     

Patterning of 293T fibroblasts on a mica surface

Controllable cell growth on the defined areas of surfaces is important for potential applications in biosensor fabrication and tissue engineering. In this study, controllable cell growth was achieved by culturing 293 T fibroblast cells on a mica surface which had been patterned with collagen strips by a microcontact printing (μCP) technique. The collagen area was designed to support cell adhesion and the native mica surface was designed to repel cell adhesion. Consequently, the resulting cell patterns should follow the micro-patterns of the collagen. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) measurement, atomic-force microscope (AFM) observation, and force-curve measurement were used to monitor property changes before and after the collagen adsorption process. Further data showed that the patterned cells were of good viability and able to perform a gene-transfection experiment in vitro. This technique should be of potential applications in the fields of biosensor fabrication and tissue engineering. Figure Controllable cells growth has been achieved by culturing 293T fibroblast cells on the mica surface which had been patterned with collagen strips by microcontact printing (μCP) technique     

表面微图案化聚乳酸膜的制备及其细胞亲和性评价

利用溶剂-非溶剂法(SNS)制备表面具有微孔图案的聚乳酸(PLA)膜和聚苯乙烯(PS)膜,并以微孔PS膜为模板,构建表面具有微岛图案的PLA膜.以此为基础,对所制备的微图案表面对PLA膜亲/疏水性及成骨细胞粘附与增殖性能的影响进行研究.结果显示微图案的存在显著增强了PLA膜的表面疏水性(水接触角90°);成骨细胞在微图案表面具有良好的铺展性,其黏附数量明显高于光滑PLA膜,但细胞的生长曲线相对较平缓,显示微图案表面虽有利于细胞在PLA膜表面的粘附与铺展,但对促进细胞的增殖无贡献.     

Cell adhesion promotion strategies for signal transduction enhancement in microelectrode array in vitro electrophysiology: An introductory overview and critical discussion

Microelectrode arrays (MEAs) find application both in vitro and in vivo to record and stimulate electrical activity in electrogenic cells such as neurons, cardiomyocytes, pancreatic beta cells or immortalized cell lines derived therefrom (e.g., PC12, HL-1). In MEA electrophysiology, the quality of the predominantly extracellularly recorded or elicited electrical signals strongly depends on the distance, strength and stability of the interfacial contact between the electrogenic cells and an electrode. Decorating the substrate or electrode with biochemical adhesion factors and physical guidance cues does not only determine the tightness of that junction, but it also modulates substrate biocompatibility, its biostability, cell differentiation as well as cell fate. If an interface is furthermore topologically, chemically or physically patterned or constrained, neural interconnectivity may be steered towards directional organization. In this introductory and selective overview, we briefly discuss adhesion events at the chemical and biological level, review the general role and mechanisms of cell adhesion in (neuro)biology, then explore how cells adhere to artificial substrates. This will lead to the discussion of popular strategies for enhancing and steering interfacial interactions at the bio-hardware boundary with particular focus on MEA substrates. It will include a critical treatment of open issues with respect to the origin and shape of extracellularly recorded signals and their modulation by cell-culture-inherent events.     

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.