Fluidic Force Microscopy and Atomic Force Microscopy Unveil New Insights into the Interactions of Preosteoblasts with 3D-Printed Submicron Patterns.

Physical patterns represent potential surface cues for promoting osteogenic differentiation of stem cells and improving osseointegration of orthopedic implants. Understanding the early cell-surface interactions and their effects on late cellular functions is essential for a rational design of such topographies, yet still elusive. In this work, fluidic force microscopy (FluidFM) and atomic force microscopy (AFM) combined with optical and electron microscopy are used to quantitatively investigate the interaction of preosteoblasts with 3D-printed patterns after 4 and 24 h of culture. The patterns consist of pillars with the same diameter (200 nm) and interspace (700 nm) but distinct heights (500 and 1000 nm) and osteogenic properties. FluidFM reveals a higher cell adhesion strength after 24 h of culture on the taller pillars (32 ± 7 kPa versus 21.5 ± 12.5 kPa). This is associated with attachment of cells partly on the sidewalls of these pillars, thus requiring larger normal forces for detachment. Furthermore, the higher resistance to shear forces observed for these cells indicates an enhanced anchorage and can be related to the persistence and stability of lamellipodia. The study explains the differential cell adhesion behavior induced by different pillar heights, enabling advancements in the rational design of osteogenic patterns.

Combining atomic force microscopy with complementary techniques for multidimensional single-cell analysis.

The advent of atomic force microscopy (AFM) provides an amazing instrument for characterising the structures and properties of living biological systems under aqueous conditions with unprecedented spatiotemporal resolution. In addition to its own unique capabilities for applications in life sciences, AFM is highly compatible and has been widely integrated with various complementary techniques to simultaneously sense the multidimensional (biological, chemical and physical) properties of biological systems, offering novel possibilities for comprehensively revealing the underlying mechanisms guiding life activities particularly in the studies of single cells. Herein, typical combinations of AFM and complementary techniques (including optical microscopy, ultrasound, infrared spectroscopy, Raman spectroscopy, fluidic force microscopy and traction force microscopy) and their applications in single-cell analysis are reviewed. The future perspectives are also provided.

Fluidic Force Microscopy Demonstrates That Homophilic Adhesion by Candida albicans Als Proteins Is Mediated by Amyloid Bonds between Cells.

The fungal pathogen Candida albicans frequently forms drug-resistant biofilms in hospital settings and in chronic disease patients. Cell adhesion and biofilm formation involve a family of cell surface Als (agglutinin-like sequence) proteins. It is now well documented that amyloid-like clusters of laterally arranged Als proteins activate cell-cell adhesion under mechanical stress, but whether amyloid-like bonds form between aggregating cells is not known. To address this issue, we measure the forces driving Als5-mediated intercellular adhesion using an innovative fluidic force microscopy platform. Strong cell-cell adhesion is dependent on expression of amyloid-forming Als5 at high cell surface density and is inhibited by a short antiamyloid peptide. Furthermore, there is greatly attenuated binding between cells expressing amyloid-forming Als5 and cells with a nonamyloid form of Als5. Thus, homophilic bonding between Als5 proteins on adhering cells is the major mode of fungal aggregation, rather than protein-ligand interactions. These results point to a model whereby amyloid-like ß-sheet interactions play a dual role in cell-cell adhesion, that is, in formation of adhesin nanoclusters ( cis-interactions) and in homophilic bonding between amyloid sequences on opposing cells ( trans-interactions). Because potential amyloid-forming sequences are found in many microbial adhesins, we speculate that this novel mechanism of amyloid-based homophilic adhesion might be widespread and could represent an interesting target for treating biofilm-associated infections.

Probing the reduction of adhesion forces between biofilms and anti-biofouling filtration membrane surfaces using FluidFM technology.

Biofouling is a persistent problem in many sectors (healthcare, medicine, marine, and membrane filtration processes). To control the biofouling of surfaces, it is essential to overcome or reduce the adhesion forces between biofilms and surfaces. To access and understand the molecular basis of these interactions, atomic force microscopy (AFM) is a well-suited technology that can measure adhesion forces at the piconewton level. However, AFM-based existing methods only probe interactions between individual cells and surfaces, which is not representative of realistic conditions given that bacteria mainly exist in biofilms. We develop here an original method using FluidFM, a combination of AFM and microfluidics, to probe the adhesion forces between biofilms and filtration membranes modified with an anti-biofouling agent, vanillin. This strategy involves i) growing bacterial biofilms on micrometer-sized polystyrene beads, ii) aspirating these biofilm beads at the aperture of microfluidic cantilevers and iii) using them as probes in force spectroscopy experiments. The results obtained first showed that COOH-functionalized polystyrene beads are more suitable for bacterial growth, and that biofilms obtained after 3 h of incubation could be used with FluidFM. Then, biofilm-scale force spectroscopy experiments showed a significant decrease in adhesion forces, adhesion work, and adhesion events after membrane modification, demonstrating the potential of vanillin-coated membranes to reduce biofouling. In addition, the comparison between results at the individual cell and biofilm scales highlighted the complexity of polymeric matrix unbinding and/or unfolding in the biofilm, showing that individual cells behave differently from biofilms. Overall, this method could have implications in the fields of materials science, chemical engineering, health, and the environment.

Evidence of collective influence in innate sensing using fluidic force microscopy.

The innate immune system initiates early response to infection by sensing molecular patterns of infection through pattern-recognition receptors (PRRs). Previous work on PRR stimulation of macrophages revealed significant heterogeneity in single cell responses, suggesting the importance of individual macrophage stimulation. Current methods either isolate individual macrophages or stimulate a whole culture and measure individual readouts. We probed single cell NF-κB responses to localized stimuli within a naïve culture with Fluidic Force Microscopy (FluidFM). Individual cells stimulated in naïve culture were more sensitive compared to individual cells in uniformly stimulated cultures. In cluster stimulation, NF-κB activation decreased with increased cell density or decreased stimulation time. Our results support the growing body of evidence for cell-to-cell communication in macrophage activation, and limit potential mechanisms. Such a mechanism might be manipulated to tune macrophage sensitivity, and the density-dependent modulation of sensitivity to PRR signals could have relevance to biological situations where macrophage density increases.

Engineering Endosymbiotic Growth of E. coli in Mammalian Cells.

Endosymbioses are cellular mergers in which one cell lives within another cell and have led to major evolutionary transitions, most prominently to eukaryogenesis. Generation of synthetic endosymbioses aims to provide a defined starting point for studying fundamental processes in emerging endosymbiotic systems and enable the engineering of cells with novel properties. Here, we tested the potential of different bacteria for artificial endosymbiosis in mammalian cells. To this end, we adopted the fluidic force microscopy technology to inject diverse bacteria directly into the cytosol of HeLa cells and examined the endosymbiont-host interactions by real-time fluorescence microscopy. Among them, Escherichia coli grew exponentially within the cytoplasm, however, at a faster pace than its host cell. To slow down the intracellular growth of E. coli, we introduced auxotrophies in E. coli and demonstrated that the intracellular growth rate can be reduced by limiting the uptake of aromatic amino acids. In consequence, the survival of the endosymbiont-host pair was prolonged. The presented experimental framework enables studying endosymbiotic candidate systems at high temporal resolution and at the single cell level. Our work represents a starting point for engineering a stable, vertically inherited endosymbiosis.

Cell-substratum and cell-cell adhesion forces and single-cell mechanical properties in mono- and multilayer assemblies from robotic fluidic force microscopy.

The epithelium covers, protects, and actively regulates various formations and cavities of the human body. During embryonic development the assembly of the epithelium is crucial to the organoid formation, and the invasion of the epithelium is an essential step in cancer metastasis. Live cell mechanical properties and associated forces presumably play an important role in these biological processes. However, the direct measurement of cellular forces in a precise and high-throughput manner is still challenging. We studied the cellular adhesion maturation of epithelial Vero monolayers by measuring single-cell force-spectra with high-throughput fluidic force microscopy (robotic FluidFM). Vero cells were grown on gelatin-covered plates in different seeding concentrations, and cell detachment forces were recorded from the single-cell state, through clustered island formation, to their complete assembly into a sparse and then into a tight monolayer. A methodology was proposed to separate cell-substratum and cell-cell adhesion force and energy (work of adhesion) contributions based on the recorded force-distance curves. For comparison, cancerous HeLa cells were also measured in the same settings. During Vero monolayer formation, a significantly strengthening adhesive tendency was found, showing the development of cell-cell contacts. Interestingly, this type of step-by-step maturation was absent in HeLa cells. The attachment of cancerous HeLa cells to the assembled epithelial monolayers was also measured, proposing a new high-throughput method to investigate the biomechanics of cancer cell invasion. We found that HeLa cells adhere significantly stronger to the tight Vero monolayer than cells of the same origin. Moreover, the mechanical characteristics of Vero monolayers upon cancerous HeLa cell influence were recorded and analyzed. All these results provide insight into the qualitative assessment of cell-substratum and cell-cell mechanical contacts in mono- and multilayered assemblies and demonstrate the robustness and speed of the robotic FluidFM technology to reveal biomechanical properties of live cell assemblies with statistical significances.

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.

Host EPAC1 Modulates Rickettsial Adhesion to Vascular Endothelial Cells via Regulation of ANXA2 Y23 Phosphorylation.

INTRODUCTION: Intracellular cAMP receptor exchange proteins directly activated by cAMP 1 (EPAC1) regulate obligate intracellular parasitic bacterium rickettsial adherence to and invasion into vascular endothelial cells (ECs). However, underlying precise mechanism(s) remain unclear. The aim of the study is to dissect the functional role of the EPAC1-ANXA2 signaling pathway during initial adhesion of rickettsiae to EC surfaces. METHODS: In the present study, an established system that is anatomically based and quantifies bacterial adhesion to ECs in vivo was combined with novel fluidic force microscopy (FluidFM) to dissect the functional role of the EPAC1-ANXA2 signaling pathway in rickettsiae-EC adhesion. RESULTS: The deletion of the EPAC1 gene impedes rickettsial binding to endothelium in vivo. Rickettsial OmpB shows a host EPAC1-dependent binding strength on the surface of a living brain microvascular EC (BMEC). Furthermore, ectopic expression of phosphodefective and phosphomimic mutants replacing tyrosine (Y) 23 of ANXA2 in ANXA2-knock out BMECs results in different binding force to reOmpB in response to the activation of EPAC1. CONCLUSIONS: EPAC1 modulates rickettsial adhesion, in association with Y23 phosphorylation of the binding receptor ANXA2. Underlying mechanism(s) should be further explored to delineate the accurate role of cAMP-EPAC system during rickettsial infection.

Fluidic Force Microscopy Captures Amyloid Bonds between Microbial Cells.

Fluidic force microscopy (FluidFM) is a recent force-controlled pipette technology that enables manipulation of single cells. FluidFM can be used for quantification of forces between single cells, and a novel mode of cell-cell adhesion was uncovered: amyloid-like interactions that mediate homophilic adhesion in the fungal pathogen Candida albicans.

Easy-to-Prepare Coating of Standard Cell Culture Dishes for Cell-Sheet Engineering Using Aqueous Solutions of Poly(2-n-propyl-oxazoline).

Cell-sheet technology is a well-known method by which cells are grown on thermoswitchable substrates that become nonadhesive upon cooling, such that a complete layer of adherent cells, along with the produced extracellular matrix, detaches as a sheet. Polymers that exhibit a lower critical solution temperature (LCST) below physiological temperature in water, commonly poly(N-isopropylacrylamide) (PNIPAM), are covalently grafted or, for block copolymers, physisorbed onto substrates in a monomolecular thin film to achieve this. Consequently, such substrates, and the polymers required for film formation, can only be prepared in a chemical lab with profound macromolecular expertise. In this study, we present an easy and robust method to coat standard cell culture dishes with aqueous solutions of commercially available poly(2-n-propyl-2-oxazoline) (PnPrOx), a polymer that exhibits LCST behavior. Different standard cell culture dishes were repeatedly coated with 0.1 wt % aqueous solutions of PnPrOx and dried in an oven to create a fully covered and thermoresponsive surface. Using this PnPrOx surface a variety of cell types including endothelial cells, mesenchymal stem cells, and fibroblasts, were seeded and cultured until confluency. By decreasing the temperature to 16 °C, viable cell sheets were detached within cell-type dependent time frames and could be harvested for biological analysis. We show that the cytoskeleton rearranges, leading to a more contracted morphology of the cells in the detached cell sheet. The cellular junctions between single cells within the sheet could be detected using immunostainings, indicating that strong and intact intracellular contacts are preserved in the harvested sheets.

Quantification of intercellular adhesion forces measured by fluid force microscopy.

The mechanics of cancer cell adhesion to its neighboring cells, homotypic or heterotypic, have significant impact on tumor progression and metastasis. Intercellular adhesion has been quantified previously using atomic force microscopy-based methods. Here we show the feasibility of the recently developed fluidic force microscopy (FluidFM) to measure adhesive forces exerted by breast cancer cells. Multiple cell pairs were assessed at precisely controlled, increasing contact durations by pressure-dependent immobilization of a cell at the probe tip. Eliminating chemical fixation of the cell at the tip ensured repeated use of the same probe and also minimized changes in cell physiology. Our data indicates distinct trends of adhesion forces between homotypic breast cancer cells compared to heterotypic adhesion between cancer-fibroblast and cancer-epithelial cell pairs. Adhesion forces were similar for all three cell pairs at short contact duration (< 1min) but differed at longer contact period (30min). Our study suggests that FluidFM is a rapid efficient technique that could be used to assess heterogeneity in cellular adhesion at various stages of malignant transformation.

Force-controlled manipulation of single cells: from AFM to FluidFM.

The ability to perturb individual cells and to obtain information at the single-cell level is of central importance for addressing numerous biological questions. Atomic force microscopy (AFM) offers great potential for this prospering field. Traditionally used as an imaging tool, more recent developments have extended the variety of cell-manipulation protocols. Fluidic force microscopy (FluidFM) combines AFM with microfluidics via microchanneled cantilevers with nano-sized apertures. The crucial element of the technology is the connection of the hollow cantilevers to a pressure controller, allowing their operation in liquid as force-controlled nanopipettes under optical control. Proof-of-concept studies demonstrated a broad spectrum of single-cell applications including isolation, deposition, adhesion and injection in a range of biological systems.