Microfluidic Approach to Resolve Simultaneous and Sequential Cytokine Secretion of Individual Polyfunctional Cells.

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. This response involves numerous immune cells secreting various cytokines to orchestrate the immune reaction. However, the secretion dynamics, amounts, and co-occurrence of the different cytokines by various cell subtypes remain poorly understood due to a lack of appropriate tools to study them. Here, we describe a protocol using a microfluidic droplet platform that allows the time-resolved quantitative measurement of secretion dynamics for several cytokines in parallel on the single-cell level. This is enabled by the encapsulation of individual cells into microfluidic droplets together with a multiplexed immunoassay for parallel quantification of cytokine concentrations, their immobilization for dynamic fluorescent imaging, and the analysis of the respective images to derive secreted quantities and dynamics. The protocol describes the preparation of functionalized magnetic nanoparticles, calibration experiments, cell preparation, and the encapsulation of the cells and nanoparticles into droplets for fluorescent imaging and subsequent image and data analysis using the example of lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The presented platform identified distinct cytokine secretion behavior for single and co-secreting cells, characterizing the expected phenotypic heterogeneity in the measured cell sample. Furthermore, the modular nature of the assay allows its adaptation and application to study a variety of proteins, cytokines, and cell samples, potentially leading to a deeper understanding of the interplay between different immune cell types and the role of the different cytokines secreted dynamically to shape the tightly regulated immune response. These new insights could be particularly interesting in the studies of immune dysregulations or in identifying target populations in therapy and drug development.

Antibodies, repertoires and microdevices in antibody discovery and characterization.

Therapeutic antibodies are paramount in treating a wide range of diseases, particularly in auto-immunity, inflammation and cancer, and novel antibody candidates recognizing a vast array of novel antigens are needed to expand the usefulness and applications of these powerful molecules. Microdevices play an essential role in this challenging endeavor at various stages since many general requirements of the overall process overlap nicely with the general advantages of microfluidics. Therefore, microfluidic devices are rapidly taking over various steps in the process of new candidate isolation, such as antibody characterization and discovery workflows. Such technologies can allow for vast improvements in time-lines and incorporate conservative antibody stability and characterization assays, but most prominently screenings and functional characterization within integrated workflows due to high throughput and standardized workflows. First, we aim to provide an overview of the challenges of developing new therapeutic candidates, their repertoires and requirements. Afterward, this review focuses on the discovery of antibodies using microfluidic systems, technological aspects of micro devices and small-scale antibody protein characterization and selection, as well as their integration and implementation into antibody discovery workflows. We close with future developments in microfluidic detection and antibody isolation principles and the field in general.

Multiscale mechanical analysis of the elastic modulus of skin.

The mechanical properties of the skin determine tissue function and regulate dermal cell behavior. Yet measuring these properties remains challenging, as evidenced by the large range of elastic moduli reported in the literature-from below one kPa to hundreds of MPa. Here, we reconcile these disparate results by dedicated experiments at both tissue and cellular length scales and by computational models considering the multiscale and multiphasic tissue structure. At the macroscopic tissue length scale, the collective behavior of the collagen fiber network under tension provides functional tissue stiffness, and its properties determine the corresponding elastic modulus (100-200 kPa). The compliant microscale environment (0.1-10 kPa), probed by atomic force microscopy, arises from the ground matrix without engaging the collagen fiber network. Our analysis indicates that indentation-based elasticity measurements, although probing tissue properties at the cell-relevant length scale, do not assess the deformation mechanisms activated by dermal cells when exerting traction forces on the extracellular matrix. Using dermal-equivalent collagen hydrogels, we demonstrate that indentation measurements of tissue stiffness do not correlate with the behavior of embedded dermal fibroblasts. These results provide a deeper understanding of tissue mechanics across length scales with important implications for skin mechanobiology and tissue engineering. STATEMENT OF SIGNIFICANCE: Measuring the mechanical properties of the skin is essential for understanding dermal cell mechanobiology and designing tissue-engineered skin substitutes. However, previous results reported for the elastic modulus of skin vary by six orders of magnitude. We show that two distinct deformation mechanisms, related to the tension-compression nonlinearity of the collagen fiber network, can explain the large variations in elastic moduli. Furthermore, we show that microscale indentation, which is frequently used to assess the stiffness perceived by cells, fails to engage the fiber network, and therefore cannot predict the behavior of dermal fibroblasts in stiffness-tunable fibrous hydrogels. This has important implications for how to measure and interpret the mechanical properties of soft tissues across length scales.

Experimental and Data Analysis Workflow for Soft Matter Nanoindentation.

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (µm). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young's Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with µm spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells. In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferrule-top optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer's guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.

Probing the interactions between air bubbles and (bio)interfaces at the nanoscale using FluidFM technology.

Understanding the molecular mechanisms underlying bubble-(bio)surfaces interactions is currently a challenge that if overcame, would allow to understand and control the various processes in which they are involved. Atomic force microscopy is a useful technique to measure such interactions, but it is limited by the large size and instability of the bubbles that it can use, attached either on cantilevers or on surfaces. We here present new developments where microsized and stable bubbles are produced using FluidFM technology, which combines AFM and microfluidics. The air bubbles produced were used to probe the interactions with hydrophobic samples, showing that bubbles in water behave like hydrophobic surfaces. They thus could be used to measure the hydrophobic properties of microorganisms' surfaces, but in this case the interactions are also influenced by electrostatic forces. Finally a strategy was developed to functionalize their surface, thereby modulating their interactions with microorganism interfaces. This new method provides a valuable tool to understand bubble-(bio)surfaces interactions but also to engineer them.

An Approach for the Real-Time Quantification of Cytosolic Protein-Protein Interactions in Living Cells.

In recent years, cell-based assays have been frequently used in molecular interaction analysis. Cell-based assays complement traditional biochemical and biophysical methods, as they allow for molecular interaction analysis, mode of action studies, and even drug screening processes to be performed under physiologically relevant conditions. In most cellular assays, biomolecules are usually labeled to achieve specificity. In order to overcome some of the drawbacks associated with label-based assays, we have recently introduced "cell-based molography" as a biosensor for the analysis of specific molecular interactions involving native membrane receptors in living cells. Here, we expand this assay to cytosolic protein-protein interactions. First, we created a biomimetic membrane receptor by tethering one cytosolic interaction partner to the plasma membrane. The artificial construct is then coherently arranged into a two-dimensional pattern within the cytosol of living cells. Thanks to the molographic sensor, the specific interactions between the coherently arranged protein and its endogenous interaction partners become visible in real time without the use of a fluorescent label. This method turns out to be an important extension of cell-based molography because it expands the range of interactions that can be analyzed by molography to those in the cytosol of living cells.

Elasticity spectra as a tool to investigate actin cortex mechanics.

BACKGROUND: The mechanical properties of single living cells have proven to be a powerful marker of the cell physiological state. The use of nanoindentation-based single cell force spectroscopy provided a wealth of information on the elasticity of cells, which is still largely to be exploited. The simplest model to describe cell mechanics is to treat them as a homogeneous elastic material and describe it in terms of the Young's modulus. Beside its simplicity, this approach proved to be extremely informative, allowing to assess the potential of this physical indicator towards high throughput phenotyping in diagnostic and prognostic applications. RESULTS: Here we propose an extension of this analysis to explicitly account for the properties of the actin cortex. We present a method, the Elasticity Spectra, to calculate the apparent stiffness of the cell as a function of the indentation depth and we suggest a simple phenomenological approach to measure the thickness and stiffness of the actin cortex, in addition to the standard Young's modulus. CONCLUSIONS: The Elasticity Spectra approach is tested and validated on a set of cells treated with cytoskeleton-affecting drugs, showing the potential to extend the current representation of cell mechanics, without introducing a detailed and complex description of the intracellular structure.

Morphogenesis Guided by 3D Patterning of Growth Factors in Biological Matrices.

Three-dimensional (3D) control over the placement of bioactive cues is fundamental to understand cell guidance and develop engineered tissues. Two-photon patterning (2PP) provides such placement at micro- to millimeter scale, but nonspecific interactions between proteins and functionalized extracellular matrices (ECMs) restrict its use. Here, a 2PP system based on nonfouling hydrophilic photocages and Sortase A (SA)-based enzymatic coupling is presented, which offers unprecedented orthogonality and signal-to-noise ratio in both inert hydrogels and complex mammalian matrices. Improved photocaged peptide synthesis and protein functionalization protocols with broad applicability are introduced. Importantly, the method enables 2PP in a single step in the presence of fragile biomolecules and cells, and is compatible with time-controlled growth factor presentation. As a corollary, the guidance of axons through 3D-patterned nerve growth factor (NGF) within brain-mimetic ECMs is demonstrated. The approach allows for the interrogation of the role of complex signaling molecules in 3D matrices, thus helping to better understand biological guidance in tissue development and regeneration.

Localized detection of ions and biomolecules with a force-controlled scanning nanopore microscope.

Proteins, nucleic acids and ions secreted from single cells are the key signalling factors that determine the interaction of cells with their environment and the neighbouring cells. It is possible to study individual ion channels by pipette clamping, but it is difficult to dynamically monitor the activity of ion channels and transporters across the cellular membrane. Here we show that a solid-state nanopore integrated in an atomic force microscope can be used for the stochastic sensing of secreted molecules and the activity of ion channels in arbitrary locations both inside and outside a cell. The translocation of biomolecules and ions through the nanopore is observed in real time in live cells. The versatile nature of this approach allows us to detect specific biomolecules under controlled mechanical confinement and to monitor the ion-channel activities of single cells. Moreover, the nanopore microscope was used to image the surface of the nuclear membrane via high-resolution scanning ion conductance measurements.

Dark-Field Microwells toward High-Throughput Direct miRNA Sensing with Gold Nanoparticles.

MicroRNA (miRNA) is a class of short RNA that is emerging as an ideal biomarker, as its expression level has been found to correlate with different types of diseases including diabetes and cancer. The detection of miRNA is highly beneficial for early diagnostics and disease monitoring. However, miRNA sensing remains difficult because of its small size and low expression levels. Common techniques such as quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridization and Northern blotting have been developed to quantify miRNA in a given sample. Nevertheless, these methods face common challenges in point-of-care practice as they either require complicated sample handling and expensive equipment, or suffer from low sensitivity. Here we present a new tool based on dark-field microwells to overcome these challenges in miRNA sensing. This miniaturized device enables the readout of a gold nanoparticle assay without the need of a dark-field microscope. We demonstrate the feasibility of the dark-field microwells to detect miRNA in both buffer solution and cell lysate. The dark-field microwells allow affordable miRNA sensing at a high throughput which make them a promising tool for point-of-care diagnostics.