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.

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.

Micropatterning of living single cells and cell clusters over millimeter–centimeter scale areas is of high demand in the development of cell-based biosensors. Micropatterning methodologies require both a suitable biomimetic support and a printing technology. In this work, we present the micropatterning of living mammalian cells on carboxymethyl dextran (CMD) hydrogel layers using the FluidFM BOT technology. In contrast to the ultrathin (few nanometers thick in the dry state) CMD films generally used in label-free biosensor applications, we developed CMD layers with thicknesses of several tens of nanometers in order to provide support for the controlled adhesion of living cells. The fabrication method and detailed characterization of the CMD layers are also described. The antifouling ability of the CMD surfaces is demonstrated by in situ optical waveguide lightmode spectroscopy measurements using serum modeling proteins with different electrostatic properties and molecular weights. Cell micropatterning on the CMD surface was obtained by printing cell adhesion mediating cRGDfK peptide molecules (cyclo­(Arg-Gly-Asp-d-Phe-Lys)) directly from aqueous solution using microchanneled cantilevers with subsequent incubation of the printed surfaces in the living cell culture. Uniquely, we present cell patterns with different geometries (spot, line, and grid arrays) covering both micrometer and millimeter–centimeter scale areas. The adhered patterns were analyzed by phase contrast microscopy and the adhesion process on the patterns was real-time monitored by digital holographic microscopy, enabling to quantify the survival and migration of cells on the printed cRGDfK arrays.

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.

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Automated manipulation and sorting of single cells are challenging, when intact cells are needed for further investigations, e.g., RNA or DNA sequencing. We applied a computer controlled micropipette on a microscope admitting 80 PCR (Polymerase Chain Reaction) tubes to be filled with single cells in a cycle. Due to the Laplace pressure, fluid starts to flow out from the micropipette only above a critical pressure preventing the precise control of drop volume in the submicroliter range. We found an anomalous pressure additive to the Laplace pressure that we attribute to the evaporation of the drop. We have overcome the problem of the critical dropping pressure with sequentially operated fast fluidic valves timed with a millisecond precision. Minimum drop volume was 0.4–0.7 μl with a sorting speed of 15–20 s per cell. After picking NE-4C neuroectodermal mouse stem cells and human primary monocytes from a standard plastic Petri dish we could gently deposit single cells inside tiny drops. 94 ± 3% and 54 ± 7% of the deposited drops contained single cells for NE-4C and monocytes, respectively. 7.5 ± 4% of the drops contained multiple cells in case of monocytes. Remaining drops were empty. Number of cells deposited in a drop could be documented by imaging the Petri dish before and after sorting. We tuned the adhesion force of cells to make the manipulation successful without the application of microstructures for trapping cells on the surface. We propose that our straightforward and flexible setup opens an avenue for single cell isolation, critically needed for the rapidly growing field of single cell biology.

CR3 and CR4, the leukocyte specific β 2-integrins, involved in cellular adherence, migration and phagocytosis, are often assumed to have similar functions. Previously however, we proved that under physiological conditions CR4 is dominant in the adhesion to fibrinogen of human monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs). Here, using inflammatory conditions, we provide further evidence that the expression and function of CR3 and CR4 are not identical in these cell types. We found that LPS treatment changes their expression differently on MDMs and MDDCs, suggesting a cell type specific regulation. Using mAb24, specific for the high affinity conformation of CD18, we proved that the activation and recycling of β 2-integrins is significantly enhanced upon LPS treatment. Adherence to fibrinogen was assessed by two fundamentally different approaches: a classical adhesion assay and a computer-controlled micropipette, capable of measuring adhesion strength. While both receptors participated in adhesion, we demonstrated that CR4 exerts a dominant role in the strong attachment of MDDCs. Studying the formation of podosomes we found that MDMs retain podosome formation after LPS activation, whereas MDDCs lose this ability, resulting in a significantly reduced adhesion force and an altered cellular distribution of CR3 and CR4. Our results suggest that inflammatory conditions reshape differentially the expression and role of CR3 and CR4 in macrophages and dendritic cells.

Reliable measurement of the binding kinetics of low molecular weight analytes to their targets is still a challenging task. Often, the introduction of labels is simply impossible in such measurements, and the application of label-free methods is the only reliable choice. By measuring the binding kinetics of Ni(II) ions to genetically modified flagellin layers, we demonstrate that: (1) Grating-Coupled Interferometry (GCI) is well suited to resolve the binding of ions, even at very low protein immobilization levels; (2) it supplies high quality kinetic data from which the number and strength of available binding sites can be determined, and (3) the rate constants of the binding events can also be obtained with high accuracy. Experiments were performed using a flagellin variant incorporating the C-terminal domain of the nickel-responsive transcription factor NikR. GCI results were compared to affinity data from titration calorimetry. We found that besides the low-affinity binding sites characterized by a micromolar dissociation constant (Kd), tetrameric FliC-NikRC molecules possess high-affinity binding sites with Kd values in the nanomolar range. GCI enabled us to obtain real-time kinetic data for the specific binding of an analyte with molar mass as low as 59 Da, even at signals lower than 1 pg/mm2.