A practical review on the measurement tools for cellular adhesion force.

Cell-cell and cell-matrix adhesions are fundamental in all multicellular organisms. They play a key role in cellular growth, differentiation, pattern formation and migration. Cell-cell adhesion is substantial in the immune response, pathogen-host interactions, and tumor development. The success of tissue engineering and stem cell implantations strongly depends on the fine control of live cell adhesion on the surface of natural or biomimetic scaffolds. Therefore, the quantitative and precise measurement of the adhesion strength of living cells is critical, not only in basic research but in modern technologies, too. Several techniques have been developed or are under development to quantify cell adhesion. All of them have their pros and cons, which has to be carefully considered before the experiments and interpretation of the recorded data. Current review provides a guide to choose the appropriate technique to answer a specific biological question or to complete a biomedical test by measuring cell adhesion.

Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA.

Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes.

CD11c/CD18 Dominates Adhesion of Human Monocytes, Macrophages and Dendritic Cells over CD11b/CD18.

Complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) belong to the family of beta2 integrins and are expressed mainly by myeloid cell types in humans. Previously, we proved that CR3 rather than CR4 plays a key role in phagocytosis. Here we analysed how CD11b and CD11c participate in cell adhesion to fibrinogen, a common ligand of CR3 and CR4, employing human monocytes, monocyte-derived macrophages (MDMs) and monocyte-derived dendritic cells (MDDCs) highly expressing CD11b as well as CD11c. We determined the exact numbers of CD11b and CD11c on these cell types by a bead-based technique, and found that the ratio of CD11b/CD11c is 1.2 for MDDCs, 1.7 for MDMs and 7.1 for monocytes, suggesting that the function of CD11c is preponderant in MDDCs and less pronounced in monocytes. Applying state-of-the-art biophysical techniques, we proved that cellular adherence to fibrinogen is dominated by CD11c. Furthermore, we found that blocking CD11b significantly enhances the attachment of MDDCs and MDMs to fibrinogen, demonstrating a competition between CD11b and CD11c for this ligand. On the basis of the cell surface receptor numbers and the measured adhesion strength we set up a model, which explains the different behavior of the three cell types.

Flagellin based biomimetic coatings: From cell-repellent surfaces to highly adhesive coatings.

UNLABELLED: Biomimetic coatings with cell-adhesion-regulating functionalities are intensively researched today. For example, cell-based biosensing for drug development, biomedical implants, and tissue engineering require that the surface adhesion of living cells is well controlled. Recently, we have shown that the bacterial flagellar protein, flagellin, adsorbs through its terminal segments to hydrophobic surfaces, forming an oriented monolayer and exposing its variable D3 domain to the solution. Here, we hypothesized that this nanostructured layer is highly cell-repellent since it mimics the surface of the flagellar filaments. Moreover, we proposed flagellin as a carrier molecule to display the cell-adhesive RGD (Arg-Gly-Asp) peptide sequence and induce cell adhesion on the coated surface. The D3 domain of flagellin was replaced with one or more RGD motifs linked by various oligopeptides modulating flexibility and accessibility of the inserted segment. The obtained flagellin variants were applied to create surface coatings inducing cell adhesion and spreading to different levels, while wild-type flagellin was shown to form a surface layer with strong anti-adhesive properties. As reference surfaces synthetic polymers were applied which have anti-adhesive (PLL-g-PEG poly(l-lysine)-graft-poly(ethylene glycol)) or adhesion inducing properties (RGD-functionalized PLL-g-PEG). Quantitative adhesion data was obtained by employing optical biochips and microscopy. Cell-adhesion-regulating coatings can be simply formed on hydrophobic surfaces by using the developed flagellin-based constructs. The developed novel RGD-displaying flagellin variants can be easily obtained by bacterial production and can serve as alternatives to create cell-adhesion-regulating biomimetic coatings. STATEMENT OF SIGNIFICANCE: In the present work, we show for the first time that.

Adhesion kinetics of human primary monocytes, dendritic cells, and macrophages: Dynamic cell adhesion measurements with a label-free optical biosensor and their comparison with end-point assays.

Monocytes, dendritic cells (DCs), and macrophages (MFs) are closely related immune cells that differ in their main functions. These specific functions are, to a considerable degree, determined by the differences in the adhesion behavior of the cells. To study the inherently and essentially dynamic aspects of the adhesion of monocytes, DCs, and MFs, dynamic cell adhesion assays were performed with a high-throughput label-free optical biosensor [Epic BenchTop (BT)] on surfaces coated with either fibrinogen (Fgn) or the biomimetic copolymer PLL-g-PEG-RGD. Cell adhesion profiles typically reached their maximum at ∼60 min after cell seeding, which was followed by a monotonic signal decrease, indicating gradually weakening cell adhesion. According to the biosensor response, cell types could be ordered by increasing adherence as monocytes, MFs, and DCs. Notably, all three cell types induced a larger biosensor signal on Fgn than on PLL-g-PEG-RGD. To interpret this result, the molecular layers were characterized by further exploiting the potentials of the biosensor: by measuring the adsorption signal induced during the surface coating procedure, the authors could estimate the surface density of adsorbed molecules and, thus, the number of binding sites potentially presented for the adhesion receptors. Surfaces coated with PLL-g-PEG-RGD presented less RGD sites, but was less efficient in promoting cell spreading than those coated with Fgn; hence, other binding sites in Fgn played a more decisive role in determining cell adherence. To support the cell adhesion data obtained with the biosensor, cell adherence on Fgn-coated surfaces 30-60 min after cell seeding was measured with three complementary techniques, i.e., with (1) a fluorescence-based classical adherence assay, (2) a shear flow chamber applying hydrodynamic shear stress to wash cells away, and (3) an automated micropipette using vacuum-generated fluid flow to lift cells up. These techniques confirmed the results obtained with the high-temporal-resolution Epic BT, but could only provide end-point data. In contrast, complex, nonmonotonic cell adhesion kinetics measured by the high-throughput optical biosensor is expected to open a window on the hidden background of the immune cell-extracellular matrix interactions.

Intensity interrogation near cutoff resonance for label-free cellular profiling.

We report a method enabling intensity-based readout for label-free cellular assays, and realize a reader device with the same footprint as a microtiter plate. For unambiguous resonance intensity measurements in resonance waveguide grating (RWG) sensors, we propose to apply resonances near the substrate cutoff wavelength. This method was validated in bulk refractive index, surface bilayer and G protein-coupled receptor (GPCR) experiments. The significantly reduced size of the reader device opens new opportunities for easy integration into incubators or liquid handling systems.

Automated single cell isolation from suspension with computer vision.

Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1-2 nanoliters from a thin (~100 µm) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any tissue cell type. Combination of 1 µm positioning precision, adaptive cell targeting and below 1 nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation. Single cells were injected either into the wells of a miniature plate with a sorting speed of 3 cells/min or into standard PCR tubes with 2 cells/min. We could isolate labeled cells also from dense cultures containing ~1,000 times more unlabeled cells by the successive application of the sorting process. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved.

Viscosity dependence of passage through a fluctuating bottleneck.

We generalize the model of a rate process involving the passage of an object through a fluctuating bottleneck. The rate of passage is considered to be proportional to a power function of the radius of the bottleneck with exponent α > 0. The fluctuations of the bottleneck are coupled to the motion of the surrounding medium and governed by Langevin dynamics. We show numerically and also explain analytically that for slow bottleneck fluctuations the long time decay rate of the process has a fractional power law dependence on the solvent viscosity with exponent α/(α + 2). The results are consistent with the experimental data on ligand binding to myoglobin, and might also be relevant to other reactions for which exponents between 0 and 1 were reported.

Single cell adhesion assay using computer controlled micropipette.

Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today's techniques typically have an extremely low throughput (5-10 cells per day). Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (∼30 min). We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a sub-population of strongly fibrinogen adherent cells appearing in macrophages and highly represented in dendritic cells, but not observed in monocytes.

Enhanced protein adsorption and cellular adhesion using transparent titanate nanotube thin films made by a simple and inexpensive room temperature process: application to optical biochips.

A new type of titanate nanotube (TNT) coating is investigated for exploitation in biosensor applications. The TNT layers were prepared from stable but additive-free sols without applying any binding compounds. The simple, fast spin-coating process was carried out at room temperature, and resulted in well-formed films around 10nm thick. The films are highly transparent as expected from their nanostructure and may, therefore, be useful as coatings for surface-sensitive optical biosensors to enhance the specific surface area. In addition, these novel coatings could be applied to medical implant surfaces to control cellular adhesion. Their morphology and structure was characterized by spectroscopic ellipsometry (SE) and atomic force microscopy (AFM), and their chemical state by X-ray photoelectron spectroscopy (XPS). For quantitative surface adhesion studies, the films were prepared on optical waveguides. The coated waveguides were shown to still guide light; thus, their sensing capability remains. Protein adsorption and cell adhesion studies on the titanate nanotube films and on smooth control surfaces revealed that the nanostructured titanate enhanced the adsorption of albumin; furthermore, the coatings considerably enhanced the adhesion of living mammalian cells (human embryonic kidney and preosteoblast).

Sample handling in surface sensitive chemical and biological sensing: a practical review of basic fluidics and analyte transport.

This paper gives an overview of the advantages and associated caveats of the most common sample handling methods in surface-sensitive chemical and biological sensing. We summarize the basic theoretical and practical considerations one faces when designing and assembling the fluidic part of the sensor devices. The influence of analyte size, the use of closed and flow-through cuvettes, the importance of flow rate, tubing length and diameter, bubble traps, pressure-driven pumping, cuvette dead volumes, and sample injection systems are all discussed. Typical application areas of particular arrangements are also highlighted, such as the monitoring of cellular adhesion, biomolecule adsorption-desorption and ligand-receptor affinity binding. Our work is a practical review in the sense that for every sample handling arrangement considered we present our own experimental data and critically review our experience with the given arrangement. In the experimental part we focus on sample handling in optical waveguide lightmode spectroscopy (OWLS) measurements, but the present study is equally applicable for other biosensing technologies in which an analyte in solution is captured at a surface and its presence is monitored. Explicit attention is given to features that are expected to play an increasingly decisive role in determining the reliability of (bio)chemical sensing measurements, such as analyte transport to the sensor surface; the distorting influence of dead volumes in the fluidic system; and the appropriate sample handling of cell suspensions (e.g. their quasi-simultaneous deposition). At the appropriate places, biological aspects closely related to fluidics (e.g. cellular mechanotransduction, competitive adsorption, blood flow in veins) are also discussed, particularly with regard to their models used in biosensing.

Dependence of cancer cell adhesion kinetics on integrin ligand surface density measured by a high-throughput label-free resonant waveguide grating biosensor.

A novel high-throughput label-free resonant waveguide grating (RWG) imager biosensor, the Epic® BenchTop (BT), was utilized to determine the dependence of cell spreading kinetics on the average surface density (v(RGD)) of integrin ligand RGD-motifs. v(RGD) was tuned over four orders of magnitude by co-adsorbing the biologically inactive PLL-g-PEG and the RGD-functionalized PLL-g-PEG-RGD synthetic copolymers from their mixed solutions onto the sensor surface. Using highly adherent human cervical tumor (HeLa) cells as a model system, cell adhesion kinetic data of unprecedented quality were obtained. Spreading kinetics were fitted with the logistic equation to obtain the spreading rate constant (r) and the maximum biosensor response (Δλmax), which is assumed to be directly proportional to the maximum spread contact area (Amax). r was found to be independent of the surface density of integrin ligands. In contrast, Δλmax increased with increasing RGD surface density until saturation at high densities. Interpreting the latter behavior with a simple kinetic mass action model, a 2D dissociation constant of 1753 ± 243 µm(-2) (corresponding to a 3D dissociation constant of ~30 µM) was obtained for the binding between RGD-specific integrins embedded in the cell membrane and PLL-g-PEG-RGD. All of these results were obtained completely noninvasively without using any labels.

In-situ and label-free optical monitoring of the adhesion and spreading of primary monocytes isolated from human blood: dependence on serum concentration levels.

Adhesion and spreading of primary monocytes isolated from human blood were monitored utilizing optical waveguide lightmode spectroscopy (OWLS); a highly sensitive label-free biosensor technique using evanescent optical waves generated at a biocompatible surface. Appropriate development on a custom built setup enabled the OWLS cuvette to be operated as a 1.5 ml mini-incubator, controlling both temperature and CO2 levels. The incubator-equipped OWLS is readily applicable for delicate and long-term studies on sensitive primary cells, demonstrated here through monitoring the serum dependence of the adhesion and spreading of human monocytes. Moreover, the custom-built setup enables the simultaneous monitoring of the position and overall width of the OWLS resonant peaks. This unique feature makes it possible to distinguish the refractive index variations induced by the adsorption of secreted material from refractive index changes provoked by cellular spreading. A definite attachment and spreading activity was observed on the substratum (glassy silica-titania), when the serum level of the culturing medium was 0.0-0.01%. Increasing serum concentration resulted in a steep fall in monocyte surface adhesion and spreading. 1.0% serum level practically abolished all spreading activity measured by OWLS, and the number of attached cells was significantly decreased, too. Serum addition to fully spread cells provoked a reduction in the cell-substratum contact area, clearly detectable by the biosensor. Cell spreading was inhibited by pre-coating the sensor surface with considerable amounts of serum proteins. These findings suggest that monocyte spreading is inhibited by the adsorption of serum biomolecules to the substratum, rather than by soluble factors present in the serum. All of these results were obtained completely noninvasively with real time monitoring; demonstrating the capabilities of OWLS to sensitively monitor the adhesion properties of immune cells isolated from human blood. The current study is, therefore, a significant step towards the application of label-free optical biosensors in medical diagnostics.

Optical anisotropy of flagellin layers: in situ and label-free measurement of adsorbed protein orientation using OWLS.

The surface adsorption of the protein flagellin was followed in situ using optical waveguide lightmode spectroscopy (OWLS). Flagellin did not show significant adsorption on a hydrophilic waveguide, but very rapidly formed a dense monolayer on a hydrophobic (silanized) surface. The homogeneous and isotropic optical layer model, which has hitherto been generally applied in OWLS data interpretation for adsorbed protein films, failed to characterize the flagellin layer, but it could be successfully modeled as an uniaxial thin film. This anisotropic modeling revealed a significant positive birefringence in the layer, suggesting oriented protein adsorption. The adsorbed flagellin orientation was further evidenced by monitoring the surface adsorption of truncated flagellin variants, in which the terminal protein regions or the central (D3) domain were removed. Without the terminal regions the protein adsorption was much slower and the resulting films were significantly less birefringent, implying that intact flagellin adsorbs on the hydrophobic surface via its terminal regions.