Cell-surface Interaction Studies Research Articles

VIBRATIONAL SPECTRUM CHARACTERIZATION OF OUTER SURFACE OF HELICOBACTER PYLORI BIOFILMS BY FUNCTIONALLY-ENHANCED DERIVATIVE SPECTROSCOPY (FEDS)

Mid-infrared spectroscopy in conjunction with Functionally-Enhanced Derivative Spectroscopy (IR+FEDS) is a powerful analytical tool for the improving of analysis of microorganism IR spectra. The objective of this work is to characterize the outer surface of two H. pylori strains by IR+FEDS. This work is a key stage for the study of cell-cell and cell-surface interactions between microorganisms, as well as, for polymicrobial biofilm characterizations where H. pylori species are involved. For that, artificial bacterial biofilms, or bacterial biolayers, were deposited on ultrafiltration cellulose membranes which were previously modified by covalent insertion of a spectral marker and used as sensing surface for analysis of bacterium biolayers. Biolayers were analyzed using an infrared spectrophotometer with ATR and data were analyzed by classic procedures and by deconvolution based on FEDS transform. It is concluded that, for correct application of technique is required a minimum amount of noise in the working spectra which can be achieved by simple smoothing algorithms; in addition, reproducibility must be warranted by the implementation of standardized protocols and the use of an appropriate number of samples. It is concluded that in addition to typical signals associated with IR spectrum of microorganisms, by FEDS, a better and more detailed description of outer membrane of H. pylori biofilms can be performed. In particular, the detecting and monitoring of cysteine-rich proteins can be satisfactorily performed by IR+FEDS.

Evaluation of cell-surface interaction using a 3D spheroid cell culture model on artificial extracellular matrices

Since decades, cell-surface interactions are studied in 2D cell culture approaches, but cells organized in 3D (spheroids) reflect the normal situation of cells in tissues much better due to intense cell-cell-contacts. Accordingly, this study aimed to prove, if spheroids could be used to study cell-surface interaction.Spheroids consisting of fibroblasts and/or osteoblasts were seeded on artificial extracellular matrices. Here, non-sulfated hyaluronan as a biological relevant compound of the extracellular matrix was chemically sulfated to different extents and co-fibrillised with collagen. The changes of the spheroid diameters and the migration distance of outgrown cells after seeding on the matrices were used as parameters to evaluate cell-surface interaction quantitatively.Fibroblast-based spheroids reacted in the initial phase of adhesion with different spheroid sizes on the contact with the matrices. In contrast, the reaction of osteoblasts was more pronounced at later time points exhibiting a decrease of the size of the spheroids with increasing sulfation degree of the matrix. The migration of the cells was impaired by increasing sulfation degree, which might be caused by an increased expression of focal adhesion relevant proteins. In summary, spheroids can be used in cell-surface interaction studies and additional analytical tools could be implemented.

The Effect of Size and Geometry of Poly(acrylamide) Brush-Based Micropatterns on the Behavior of Cells.

In this study, the fabrication, detailed characterization, and application of long-term stable micropatterned bio-interfaces of passivating poly(acrylamide) (PAAm) brushes on transparent gold for application in the study of cell-surface interactions is reported. The micropatterns were fabricated by microcontact printing of an initiator for surface-initiated atom transfer radical polymerization (SI-ATRP), SI-ATRP of acrylamide, and subsequently backfilling of the unfunctionalized areas of 400-2500 μm(2) size and systematically altered number of corners with octadecanethiol. As verified by surface plasmon resonance spectroscopy, the physisorption of fibronectin (FN) was restricted to the adhesive areas. Exploiting this platform, the effect of micropattern geometry and size of cell-adhesive FN areas surrounded by passivating PAAm brushes on transparent gold substrates on the attachment of cells and cytoskeleton alignment was investigated at the single-cell level. Exceptional long-term stability of the patterned PAAm brushes and arrays of adhesive areas, in which human pancreatic tumor cells (Patu 8988T) and fibroblast cells (NIH 3T3) were confined for more than one week, was observed. Adhesive areas of 1600 μm(2) or less constrained the cell shape and caused focal adhesions to accumulate in the corners of the pattern. These changes were most obvious for the PatuT cells in adhesive areas of ∼900 μm(2), in which the actin filaments were aligned, following the boundary of the pattern, and merged in the focal adhesions concentrated in the corners of the pattern. NIH 3T3 cells possessed a larger cell area, which led to an optimal cytoskeleton alignment in adhesive patterns of ∼1600 μm(2). The alignment of the cytoskeleton was found to be less pronounced in cells on larger adhesive areas, where the PatuT cells spread similarly to cells on unpatterned substrates. By contrast, the NIH 3T3 cells were found to stretch even on larger adhesive areas, spanning from one corner to the other. The long-term stability under cell culture conditions of the patterns introduced here will also be useful for long-term studies of single and multiple cells, cell motility in toxicity assays, and stem cell differentiation.

Aquatic flower-inspired cell culture platform with simplified medium exchange process for facilitating cell-surface interaction studies.

Establishing fundamentals for regulating cell behavior with engineered physical environments, such as topography and stiffness, requires a large number of cell culture experiments. However, cell culture experiments in cell-surface interaction studies are generally labor-intensive and time-consuming due to many experimental tasks, such as multiple fabrication processes in sample preparation and repetitive medium exchange in cell culture. In this work, a novel aquatic flower-inspired cell culture platform (AFIP) is presented. AFIP aims to facilitate the experiments on the cell-surface interaction studies, especially the medium exchange process. AFIP was devised to capture and dispense cell culture medium based on interactions between an elastic polymer substrate and a liquid medium. Thus, the medium exchange can be performed easily and without the need of other instruments, such as a vacuum suction and pipette. An appropriate design window of AFIP, based on scaling analysis, was identified to provide a criterion for achieving stability in medium exchange as well as various surface characteristics of the petal substrates. The developed AFIP, with physically engineered petal substrates, was also verified to exchange medium reliably and repeatedly. A closed structure capturing the medium was sustained stably during cell culture experiments. NIH3T3 proliferation results also demonstrated that AFIP can be applied to the cell-surface interaction studies as an alternative to the conventional method.

Enhanced osteogenic fate and function of MC3T3-E1 cells on nanoengineered polystyrene surfaces with nanopillar and nanopore arrays

During in vitro culture, cell fate and function, including cell adhesion, morphology, proliferation and differentiation, are affected by surface characteristics, such as geometry, wettability, hardness, chemistry and charge. This study replicated two different types of nanoengineered polystyrene surfaces (NPS) containing nanopillar (NPS-Pi) or nanopore (NPS-Po) arrays by hot embossing and investigated their topographical effects on cell behavior using osteoblast-like MC3T3-E1 cells. To mass-replicate NPS, rigid metal nano-stamps were manufactured by nickel electroforming onto two different nano-templates: (1) a nanopore-arrayed anodic aluminum oxide nano-template using two-step electrochemical oxidation and (2) a nanopillar-arrayed polymer using hot embossing process. The physical and mechanical properties of the NPS, including geometry, wettability, hardness and elastic modulus, were evaluated with the help of field emission-scanning electron microscopy, a contact angle meter, and a nanoindenter. The nanotopography maintained the bulk property, while drastically changing the surface properties. In vitro the NPS had significant effects on MC3T3-E1 cell morphology, attachment, proliferation and osteogenic differentiation compared to a flat substrate due to the altered physical and mechanical surface properties of the nanoengineered surface. Interestingly, the NPS-Po was more effective at enhancing cell proliferation and osteogenesis differentiation. One potential explanation for these results may be that the subcellular binding sites induced by the nanostructures changed the cell morphology and promoted contractile cytoskeletons, thereby enhancing osteogenic differentiation. This, which allows for the cost-effective replication of NPS and the control of cell behavior, has various applications with respect to biomedical and cell surface interaction studies, in addition to enhanced osteogenic cell fate and function.

Silk Film Culture System for <em>in vitro</em> Analysis and Biomaterial Design

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.

Silk Film Culture System for <em>in vitro</em> Analysis and Biomaterial Design

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.

Circular, nanostructured and biofunctionalized hydrogel microchannels for dynamic cell adhesion studies

We report on a method to fabricate biofunctionalized polyethylene glycol hydrogel microchannels with adjustable circular cross-sections. The inner channel surfaces are decorated with Au-nanoparticle arrays of tunable density. These Au-nanoparticles are functionalized with biomolecules whereas the hydrogel material provides an inert and biocompatible background. This technology provides control over flow conditions, channel curvature and biomolecule density on the channel surface. It can be applied for biophysical studies of cell-surface interactions mimicking, for example, leukocyte interactions with the endothelial lining in small vessels.

Scanning Thermal Lithography as a Tool for Highly Localized Nanoscale Chemical Surface Functionalization

ABSTRACTWe report on Scanning Thermal Lithography (SThL), a recently introduced lithographic tool, for local thermochemistry on tert-butyl acrylate based polymer films featuring chemical cross links. The tailored polymer films afford platforms for controlled high molecular density coupling and surface immobilization of biologically relevant molecules, such as proteins. The thermally labile tert-butyl ester groups in tert-butyl acrylate based polymer films can be cleaved in air at temperatures above 150 °C to yield carboxylic acid functional groups for further (bio)- conjugation. The films were optimized to avoid plastic deformation at the elevated temperatures used during SThL. Exploiting these properties patterns with length scales as small as 35 ± 6 nm have been successfully thermally activated with SThL. Hence SThL comprises an attractive approach for the development of e.g. (bio)sensors and platforms for cell surface interaction studies with nanoscale patterns.

A Nanoscale Interface Improves Attachment of Cast Polymers to Glass

A novel interface was prepared on glass slides that stabilizes several cast polymers against delamination under conditions necessary for the study of cell surface interactions. This interface was synthesized by deposition of zirconium tetra(tert-butoxide) from the vapor phase onto the glass followed by mild thermolysis, which gives a surface-bound zirconium oxide coating. This oxide coating improved attachment of polymer coatings cast from formic acid or methylene chloride. Nylon, polyurethane, and polyhydroxybutyrate/polyhydroxyvalerate coatings were stable against delamination from the oxide-coated glass following sonication in ethanol for more than 30 min or immersion in water at pH 8 for at least 48 h.

Micropatterning functional groups on polymer surfaces via masked vapor-phase photografting

Controlling cell behavior on biomaterial surface is the ultimate goal of cell and tissue engineering. Fabrication of biomaterials with alternatively hydrophilic/hydrophobic surface of parallel nanopatterned groves can provide biomaterial surfaces for the study of cell-surface interactions. In the present communication, masked vapor-phase photografting was used in patterning functional groups on flat polymeric substrates using poly (dimethylsiloxane) (PDMS) channels. Surface patterns were fabricated by UV-initiated photografting in the presence of a patterned PDMS mask. The approach is exemplified by patterning maleic anhydride (MAH) and acrylamide (AAm) onto poly (methyl methacrylate) (PMMA). The method offers another means to chemically functionalize and pattern polymer surface at the same time.

Inverted Microcontact Printing on Polystyrene-block-Poly(tert-butyl acrylate) Films: A Versatile Approach to Fabricate Structured Biointerfaces Across the Length Scales

The combination of the recently introduced soft lithographic technique of inverted microcontact printing (i-muCP) and spin-coated films of polystyrene- block-poly( tert-butyl acrylate) (PS 690- b-P tBA 1210) as a reactive platform is shown to yield a versatile approach for the facile fabrication of topographically structured and chemically patterned biointerfaces with characteristic spacings and distances that cross many orders of magnitude. The shortcomings of conventional muCP in printing of small features with large spacings, due to the collapse of small or high aspect ratio stamp structures, are circumvented in i-muCP by printing reactants using a featureless elastomeric stamp onto a topographically structured reactive polymer film. Prior to molecular transfer, the substrate-supported PS 690- b-P tBA 1210 films were structured by imprint lithography resulting in lateral and vertical feature sizes between >50 microm-150 nm and >1.0 microm-18 nm, respectively. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and water contact angle measurements provided evidence for the absence of surface chemical transformations during the imprinting step. Following the previously established hydrolysis and activation protocol with trifluoroacetic acid and N-hydroxysuccinimide, amino end-functionalized poly(ethylene glycol) (PEG-NH 2), as well as bovine serum albumin and fibronectin as model proteins, were successfully transferred by i-muCP and coupled covalently. As shown, i-muCP yields increased PEG coverages and thus improved performance in suppressing nonspecific adsorption of proteins by exploiting the high local concentrations in the micro- and nanocontacts during molecular transfer. The i-muCP strategy provides access to versatile biointerface platforms patterned across the length scales, as shown for guided cancer cell adhesion, which opens the pathway for systematic cell-surface interaction studies.

MEASURING CELL ADHESION FORCES AS A FUNCTION OF THE CELL CYCLE BY FORCE SPECTROSCOPY

The study of cell-surface interactions is important for several different applications, notably that of medical implants. While the effects of surfaces on cell proliferation, migration, growth and genes expression are accessible via traditional microscopy and labeling techniques, the determination of cell adhesion remains a major challenge. In the context of the European project Newbone, CSEM is studying osteosarcoma cell adhesion forces on load-bearing fiber-reinforced non-metallic bone implant materials. Atomic force microscopy, and particularly force spectroscopy are used to directly measure the cell adhesion to the surface. The adhesion of cells on surfaces depends very strongly on the phase of the cell cycle (G0, G1, S, G2 or M). In order to obtain meaningful results, it is necessary to study cell/surface adhesion for each phase separately.

An Acoustically Driven Microliter Flow Chamber on a Chip (μFCC) for Cell–Cell and Cell–Surface Interaction Studies

A novel method for pumping very small volumes of liquid by using surface acoustic waves is employed to create a microfluidic flow chamber on a chip. It holds a volume of only a few mul and its planar design provides complete architectural freedom. This allows for the reconstruction of even complex flow scenarios (e.g. curvatures, bifurcations and stenosis). Addition of polymer walls to the planar fluidic track enables cell culturing on the chip surface and the investigation of cell-cell adhesion dynamics under flow. We demonstrate the flexibility of the system for application in many areas of microfluidic investigations including blood clotting phenomena under various flow conditions and the investigation of different stages of cell adhesion.

Directional Alignment of MG63 Cells on Polymer Surfaces Containing Point Microstructures

MG63 cells cultured on regular arrays of point microstructures (posts and holes) are shown to preferentially align at certain angles to the pattern of the structures, at 0 degrees, 30 degrees, and 45 degrees in particular. The effect is found to be more pronounced for post rather than hole structures (although no significant difference is found for the angles the cells make to the holes or posts) and is thought to be due to the fact that the cells use the posts as anchorage points to hold themselves to the surface. It is also shown that cells preferentially align with the structures depending on the dimensions of the structures and the distance between neighboring structures. This is important when designing structured surfaces for cell-surface interaction studies for materials to be used in, for example, drug delivery or tissue engineering.

Tailored interfaces for biosensors and cell-surface interaction studies via activation and derivatization of polystyrene- block-poly( tert-butyl acrylate) thin films

Thin spin-coated films of polystyrene- block-poly( tert-butyl acrylate) (PS 690-b-PtBA 1210) on various substrates are introduced as versatile, robust reactive platform for the immobilization of (bio)molecules for the fabrication of tailored biointerfaces. The films are characterized by high stability and (bio)reactivity due to the presence of a glassy PS and a reactive PtBA block, respectively. The selective deprotection of the tert-butyl-ester groups in the PtBA skin layer by hydrolysis under acidic conditions, the activation with N-hydroxysuccinimide and the subsequent derivatization with amino functionalized (bio)molecules were investigated. Based on contact angle, FTIR spectroscopy and XPS, fluorescence microscopy and AFM data, it was shown that the (bio)molecules were coupled covalently to the polymer films and that high molecular coverages up to ∼2.4 poly(ethylene glycol) (PEG) molecules per nm 2 ( M n = 500 g/mol) were obtained. Organic dyes, polyamidoamine dendrimers, polypeptides, proteins and amino end-functionalized DNA were efficiently and homogeneously immobilized on the PS-PtBA platforms. Grafting of ω-amino functionalized PEG afforded surfaces with substantially reduced non-specific adsorption of proteins and DNA. Owing to the glassy nature of PS and the covalent amide linkages, the derivatized films showed excellent stability under a broad range of processing conditions. Finally, the viability of PS 690-b-PtBA 1210 platforms as versatile biointerfaces was demonstrated in DNA hybridization experiments, as well as cell-surface interaction studies using pancreatic cancer and K562 cells.

In Vitro Cell Response to a Polymer Surface Micropatterned by Laser Interference Lithography

This presentation will introduce laser interference lithography to prepare a periodic line and point micropatterns for study of cell-surface interactions. This process provides a straightforward micropatterning technique based on selective laser ablation of polymers utilizing the periodic energy distribution of two or more beam interference patterns. The micropatterns were characterized by atomic force microscopy, while the surface chemical modification was analyzed using X-ray photoelectron spectroscopy. Human pulmonary fibroblasts cultured on the surface of polycarbonate bearing line micropatterns were elongated, spindlelike, and oriented themselves along the line patterns with all different groove widths. In contrast, cells cultured on point patterns were also bipolar but showed no orientation. Further investigations demonstrated that human pulmonary fibroblast cells cultured on line and point micropatterns showed inflammatory response.

A Combined Photolithographic and Molecular‐Assembly Approach to Produce Functional Micropatterns for Applications in the Biosciences

AbstractChemical patterns have attracted substantial interest for applications in the field of biosensors, fundamental cell–surface interaction studies, tissue engineering, and biomaterials. A novel micropatterning technique is proposed here that combines a top–down approach based on photolithography and a bottom–up strategy through self‐organization of multifunctional molecules. The development of the molecular‐assembly patterning by lift‐off (MAPL) has been driven by the need to economically produce patches incorporating a controlled surface density of bioligands while inhibiting non‐specific adsorption. In the MAPL process, a photoresist pattern is transferred into the desired biochemical pattern by means of spontaneous adsorption of biologically relevant species and photoresist lift‐off. The surface between the interactive patches is subsequently rendered non‐fouling through immobilization of a polycationic poly(ethylene glycol) (PEG)‐graft polymer. We demonstrate that surface density of biotin molecules inside adhesive islands can be tailored quantitatively and that cells grow selectively on cell‐adhesive peptide patterns. MAPL is considered to be a valuable addition to the toolbox of soft‐lithography techniques for life‐science applications combining simplicity (no clean‐room equipment needed), cost‐effectiveness, reproducibility on the scale of whole wafer surfaces, and flexibility in terms of pattern geometry, chemistry, and substrate choice.

Metabolic Delivery of Ketone Groups to Sialic Acid Residues: APPLICATION TO CELL SURFACE GLYCOFORM ENGINEERING

The development of chemical strategies for decorating cells with defined carbohydrate epitopes would greatly facilitate studies of carbohydrate-mediated cell surface interactions. This report describes a general strategy for engineering the display of chemically defined oligosaccharides on cell surfaces that combines the concepts of metabolic engineering and selective chemical reactivity. Using a recently described method (Mahal, L. K., Yarema, K. J., and Bertozzi, C. R. (1997) Science 276, 1125-1128), we delivered a uniquely reactive ketone group to endogenous cell surface sialic acid residues by treating cells with the ketone-bearing metabolic precursor N-levulinoylmannosamine (ManLev). The ketone undergoes highly selective condensation reactions with complementary nucleophiles such as aminooxy and hydrazide groups. The detailed quantitative parameters of ManLev metabolism in human and nonhuman-derived cell lines were determined to establish a foundation for the modification of cell surfaces with novel epitopes at defined cell-surface densities. Ketones within the glycoconjugates on ManLev-treated cells were then reacted with synthetic aminooxy and hydrazide-functionalized carbohydrates. The remodeled cells were endowed with novel lectin binding profiles as determined by flow cytometry analysis. The simplicity and generality of this method make it well suited for use in the study of carbohydrate-mediated cell surface interactions.

Selection of carbohydrate-binding cell phenotypes using oligosaccharide-coated magnetic particles.

Neoglycoconjugate coated magnetic beads were assessed for their ability to selectively isolate human cells with known anti-carbohydrate reactivity. Four lung cancer cell lines, NCI-H146, NCI-N417D, SKMES-1, EKVX; two acute lymphoblastic leukemia lines, MOLT-4 and CCRF-CEM; and the anti- Le(c) (isolactosamine) hybridoma, LU-BCRU-G7, were tested. The neoglycoconjugates (biotinylated pseudopolysaccharides) bound uniformly to streptavidin coated magnetic beads as demonstrated by FITC labeled lectin. Streptavidin beads alone did not bind to any of the cell types. The anti- Le(c) hybridoma cell line, LU BCRU-G7, demonstrated binding only to Le(c) pseudopolysaccharide coated magnetic beads. Subsequent incubation in the presence of unlabeled pseudopolysaccharide resulted in the release of the beads from the cell surface. Although there was some heterogeneity within the individual lung and leukemic cell lines, positive cells showed strong rosette formation with the coated beads. The Adi disaccharide coated beads showed binding in all four lung cancer cell lines, with the Le(c) and the H (type1) pseudopolysaccharide-bead conjugates only reactive in the N417 and H146 SCLC lines. The range of L-selectin ligand-coated beads were all successful in binding to the acute lymphoblastic leukemia cell lines MOLT4 and CCRF-CEM. This approach provides a versatile model for the study of cell-surface carbohydrate interactions that should find application in many areas of cell biology.