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 (vRGD) of integrin ligand RGD-motifs. vRGD 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.

The interaction of the anti-adhesive coating, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its Arg-Gly-Asp (RGD) functionalized form, PLL-g-PEG-RGD, with the green tea polyphenol, epigallocatechin-gallate (EGCg) was in situ monitored. After, the kinetics of cellular adhesion on the EGCg exposed coatings were recorded in real-time. The employed plate-based waveguide biosensor is applicable to monitor small molecule binding and sensitive to sub-nanometer scale changes in cell membrane position and cell mass distribution; while detecting the signals of thousands of adhering cells. The combination of this remarkable sensitivity and throughput opens up new avenues in testing complicated models of cell-surface interactions. The systematic studies revealed that, despite the reported excellent antifouling properties of the coatings, EGCg strongly interacted with them, and affected their cell adhesivity in a concentration dependent manner. Moreover, the differences between the effects of the fresh and oxidized EGCg solutions were first demonstrated. Using a semiempirical quantumchemical method we showed that EGCg binds to the PEG chains of PLL-g-PEG-RGD and effectively blocks the RGD sites by hydrogen bonds. The calculations supported the experimental finding that the binding is stronger for the oxidative products. Our work lead to a new model of polyphenol action on cell adhesion ligand accessibility and matrix rigidity.

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.

Herbs and traditional medicines have been applied for thousands of years, but researchers started to study their mode of action at the molecular, cellular and tissue levels only recently. Nowadays, just like in ancient times, natural compounds are still determining factors in remedies. To support this statement, the recently won Nobel Prize for an anti-malaria agent from the plant sweet wormwood, which had been used to effectively treat the disease, could be mentioned. Among natural compounds and traditional Chinese medicines, the green tea polyphenol epigallocatechin gallate (EGCg) is one of the most studied active substances. In the present review, we summarize the molecular scale interactions of proteins and EGCg with special focus on its limited stability and antioxidant properties. We outline the observed biophysical effects of EGCg on various cell lines and cultures. The alteration of cell adhesion, motility, migration, stiffness, apoptosis, proliferation as well as the different impacts on normal and cancer cells are all reviewed. We also handle the works performed using animal models, microbes and clinical trials. Novel ways to develop its utilization for therapeutic purposes in the future are discussed too, for instance, using nanoparticles and green tea polyphenols together to cure illnesses and the combination of EGCg and anticancer compounds to intensify their effects. The limitations of the employed experimental models and criticisms of the interpretation of the obtained experimental data are summarized as well.

Single-cell adhesion force plays a crucial role in biological sciences, however its in-depth investigation is hindered by the extremely low throughput and the lack of temporal resolution of present techniques. While atomic force microcopy (AFM) based methods are capable of directly measuring the detachment force values between individual cells and a substrate, their throughput is limited to few cells per day, and cannot provide the kinetic evaluation of the adhesion force over the timescale of several hours. In this study a high spatial and temporal resolution resonant waveguide grating based label-free optical biosensor was combined with robotic fluidic force microscopy to monitor the adhesion of living cancer cells. In contrast to traditional fluidic force microscopy methods with a manipulation range in the order of 300-400 micrometers, the robotic device employed here can address single cells over mm-cm scale areas. This feature significantly increased measurement throughput, and opened the way to combine the technology with the employed microplate-based, large area biosensor. After calibrating the biosensor signals with the direct force measuring technology on 30 individual cells, the kinetic evaluation of the adhesion force and energy of large cell populations was performed for the first time. We concluded that the distribution of the single-cell adhesion force and energy can be fitted by lognormal functions as cells are spreading on the surface and revealed the dynamic changes in these distributions. The present methodology opens the way for the quantitative assessment of the kinetics of single-cell adhesion force and energy with an unprecedented throughput and time resolution, in a completely non-invasive manner.

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.

The glycocalyx is thought to perform a potent, but not yet defined function in cellular adhesion and signaling. Since 95% of cancer cells have altered glycocalyx structure, this role can be especially important in cancer development and metastasis. The glycocalyx layer of cancer cells directly influences cancer progression, involving the complicated kinetic process of cellular adhesion at various levels. In the present work, we investigated the effect of enzymatic digestion of specific glycocalyx components on cancer cell adhesion to RGD (arginine–glycine–aspartic acid) peptide motif displaying surfaces. High resolution kinetic data of cell adhesion was recorded by the surface sensitive label-free resonant waveguide grating (RWG) biosensor, supported by fluorescent staining of the cells and cell surface charge measurements. We found that intense removal of chondroitin sulfate (CS) and dermatan sulfate chains by chondroitinase ABC reduced the speed and decreased the strength of adhesion of HeLa cells. In contrast, mild digestion of glycocalyx resulted in faster and stronger adhesion. Control experiments on a healthy and another cancer cell line were also conducted, and the discrepancies were analysed. We developed a biophysical model which was fitted to the kinetic data of HeLa cells. Our analysis suggests that the rate of integrin receptor transport to the adhesion zone and integrin-RGD binding is strongly influenced by the presence of glycocalyx components, but the integrin-RGD dissociation is not. Moreover, based on the kinetic data we calculated the dependence of the dissociation constant of integrin-RGD binding on the enzyme concentration. We also determined the dissociation constant using a 2D receptor binding model based on saturation level static data recorded at surfaces with tuned RGD densities. We analyzed the discrepancies of the kinetic and static dissociation constants, further illuminating the role of cancer cell glycocalyx during the adhesion process. Altogether, our experimental results and modelling demonstrated that the chondroitin sulfate and dermatan sulfate chains of glycocalyx have an important regulatory function during the cellular adhesion process, mainly controlling the kinetics of integrin transport and integrin assembly into mature adhesion sites. Our results potentially open the way for novel type of cancer treatments affecting these regulatory mechanisms of cellular glycocalyx.

Functionalized nanoparticles (NPs) can penetrate into living cells and vesicles, opening up an extensive range of novel directions. For example, NPs are intensively employed in targeted drug delivery and biomedical imaging. However, the real-time kinetics and dynamics of NP-living cell interactions remained uncovered. In this study, we in situ monitored the cellular uptake of gold NPs-functionalized with positively charged alkaline thiol-into surface-adhered cancer cells, by using a high-throughput label-free optical biosensor employing resonant waveguide gratings. The characteristic kinetic curves upon NP exposure of cell-coated biosensor surfaces were recorded and compared to the kinetics of NP adsorption onto bare sensor surfaces. We demonstrated that from the above kinetic information, one can conclude about the interactions between the living cells and the NPs. Real-time biosensor data suggested the cellular uptake of the functionalized NPs by an active process. It was found that positively charged particles penetrate into the cells more effectively than negatively charged control particles, and the optimal size for the cellular uptake of the positively charged particles is around 5 nm. These conclusions were obtained in a cost-effective, fast, and high-throughput manner. The fate of the NPs was further revealed by electron microscopy on NP-exposed and subsequently fixed cells, well confirming the results obtained by the biosensor. Moreover, an ultrastructural study demonstrated the involvement of the endosomal-lysosomal system in the uptake of functionalized NPs and suggested the type of the internalization pathway.