Sounding a New Era in Biomechanics with Acoustic Force Spectroscopy.

The acoustic force spectroscopy (AFS) tool was recently introduced as a novel tool for probing mechanical properties of biomolecules, expanding the application of sound waves to high-throughput quantification of the mechanical properties of single cells. By using controlled acoustic forces in the piconewton to nanonewton range, tens to hundreds of cells functionalized by attached microspheres can simultaneously be stretched and tracked in real-time with sub millisecond time response. Since its first application, several studies have demonstrated the potential and versatility of the AFS for high-throughput measurements of force-induced molecular mechanisms, revealing insight into cellular biomechanics and mechanobiology at the molecular level. In this chapter, we describe the operation of the AFS starting with the underlying physical principles, followed by a run-down of experimental considerations, and finally leading to applications in molecular and cellular biology.

Arterial dissections: Common features and new perspectives.

Arterial dissections, which involve an abrupt tear in the wall of a major artery resulting in the intramural accumulation of blood, are a family of catastrophic disorders causing major, potentially fatal sequelae. Involving diverse vascular beds, including the aorta or coronary, cervical, pulmonary, and visceral arteries, each type of dissection is devastating in its own way. Traditionally they have been studied in isolation, rather than collectively, owing largely to the distinct clinical consequences of dissections in different anatomical locations - such as stroke, myocardial infarction, and renal failure. Here, we review the shared and unique features of these arteriopathies to provide a better understanding of this family of disorders. Arterial dissections occur commonly in the young to middle-aged, and often in conjunction with hypertension and/or migraine; the latter suggesting they are part of a generalized vasculopathy. Genetic studies as well as cellular and molecular investigations of arterial dissections reveal striking similarities between dissection types, particularly their pathophysiology, which includes the presence or absence of an intimal tear and vasa vasorum dysfunction as a cause of intramural hemorrhage. Pathway perturbations common to all types of dissections include disruption of TGF-ß signaling, the extracellular matrix, the cytoskeleton or metabolism, as evidenced by the finding of mutations in critical genes regulating these processes, including LRP1, collagen genes, fibrillin and TGF-ß receptors, or their coupled pathways. Perturbances in these connected signaling pathways contribute to phenotype switching in endothelial and vascular smooth muscle cells of the affected artery, in which their physiological quiescent state is lost and replaced by a proliferative activated phenotype. Of interest, dissections in various anatomical locations are associated with distinct sex and age predilections, suggesting involvement of gene and environment interactions in disease pathogenesis. Importantly, these cellular mechanisms are potentially therapeutically targetable. Consideration of arterial dissections as a collective pathology allows insight from the better characterized dissection types, such as that involving the thoracic aorta, to be leveraged to inform the less common forms of dissections, including the potential to apply known therapeutic interventions already clinically available for the former.

Biomechanical Characterization of Endothelial Cells Exposed to Shear Stress Using Acoustic Force Spectroscopy.

Characterizing mechanical properties of cells is important for understanding many cellular processes, such as cell movement, shape, and growth, as well as adaptation to changing environments. In this study, we explore the mechanical properties of endothelial cells that form the biological barrier lining blood vessels, whose dysfunction leads to development of many cardiovascular disorders. Stiffness of living endothelial cells was determined by Acoustic Force Spectroscopy (AFS), by pull parallel multiple functionalized microspheres located at the cell-cell periphery. The unique configuration of the acoustic microfluidic channel allowed us to develop a long-term dynamic culture protocol exposing cells to laminar flow for up to 48 h, with shear stresses in the physiological range (i.e., 6 dyn/cm2). Two different Endothelial cells lines, Human Aortic Endothelial Cells (HAECs) and Human Umbilical Vein Endothelial Cells (HUVECs), were investigated to show the potential of this tool to capture the change in cellular mechanical properties during maturation of a confluent endothelial monolayer. Immunofluorescence microscopy was exploited to follow actin filament rearrangement and junction formation over time. For both cell types we found that the application of shear-stress promotes the typical phenotype of a mature endothelium expressing a linear pattern of VE-cadherin at the cell-cell border and actin filament rearrangement along the perimeter of Endothelial cells. A staircase-like sequence of increasing force steps, ranging from 186 pN to 3.5 nN, was then applied in a single measurement revealing the force-dependent apparent stiffness of the membrane cortex in the kPa range. We also found that beads attached to cells cultured under dynamic conditions were harder to displace than cells cultured under static conditions, showing a stiffer membrane cortex at cell periphery. All together these results demonstrate that the AFS can identify changes in cell mechanics based on force measurements of adherent cells under conditions mimicking their native microenvironment, thus revealing the shear stress dependence of the mechanical properties of neighboring endothelial cells.

An Acoustic Platform for Single-Cell, High-Throughput Measurements of the Viscoelastic Properties of Cells.

Cellular processes including adhesion, migration, and differentiation are governed by the distinct mechanical properties of each cell. Importantly, the mechanical properties of individual cells can vary depending on local physical and biochemical cues in a time-dependent manner resulting in significant inter-cell heterogeneity. While several different methods have been developed to interrogate the mechanical properties of single cells, throughput to capture this heterogeneity remains an issue. Here, single-cell, high-throughput characterization of adherent cells is demonstrated using acoustic force spectroscopy (AFS). AFS works by simultaneously, acoustically driving tens to hundreds of silica beads attached to cells away from the cell surface, allowing the user to measure the stiffness of adherent cells under multiple experimental conditions. It is shown that cells undergo marked changes in viscoelasticity as a function of temperature, by altering the temperature within the AFS microfluidic circuit between 21 and 37 °C. In addition, quantitative differences in cells exposed to different pharmacological treatments specifically targeting the membrane-cytoskeleton interface are shown. Further, the high-throughput format of the AFS is utilized to rapidly probe, in excess of 1000 cells, three different cell lines expressing different levels of a mechanosensitive protein, Piezo1, demonstrating the ability to differentiate between cells based on protein expression levels.

Antibody Printing Technologies.

Antibody microarrays are routinely employed in the lab and in the clinic for studying protein expression, protein-protein, and protein-drug interactions. The microarray format reduces the size scale at which biological and biochemical interactions occur, leading to large reductions in reagent consumption and handling times while increasing overall experimental throughput. Specifically, antibody microarrays, as a platform, offer a number of different advantages over traditional techniques in the areas of drug discovery and diagnostics. While a number of different techniques and approaches have been developed for creating micro and nanoscale antibody arrays, issues relating to sensitivity, cost, and reproducibility persist. The aim of this review is to highlight current state-of the-art techniques and approaches for creating antibody arrays by providing latest accounts of the field while discussing potential future directions.

Characterization and differential retention of Q beta bacteriophage virus-like particles using cyclical electrical field-flow fractionation and asymmetrical flow field-flow fractionation.

Virus-like particles (VLPs) are widely used in medicine, but can be difficult to characterize and isolate from aggregates. In this research, primarily cyclical electrical field-flow fractionation (CyElFFF) coupled with multi-angle light scattering (MALS), and dynamic light scattering (DLS) detectors, was used for the first time to perform size and electrical characterization of three different types of Q beta bacteriophage virus-like particles (VLPs): a blank Q beta bacteriophage which is denoted as VLP and two conjugated ones with different peptides. The CyElFFF results were verified with transmission electron microscopy (TEM). Asymmetrical flow field-flow fractionation (AF4) coupled with MALS was also applied using conditions similar to those used in the CyElFFF experiments, and the results of the two techniques were compared to each other. Using these techniques, the size and electrophoretic characteristics of the fractionated VLPs in CyElFFF were obtained. The results indicate that CyElFFF can be used to obtain a clear distribution of electrophoretic mobilities for each type of VLP. Accordingly, CyElFFF was able to differentially retain and isolate VLPs with high surface electric charge/electrophoretic mobility from the ones with low electric charge/electrophoretic mobility. Regarding the size characterization, the size distribution of the eluted VLPs was obtained using both techniques. CyElFFF was able to identify subpopulations that did not appear in the AF4 results by generating a shoulder peak, whereas AF4 produced a single peak. Different size characteristics of the VLPs appearing in the shoulder peak and the main peak indicate that CyElFFF was able to isolate aggregated VLPs from the monomers partially. Graphical abstract.

Cell membrane mechanics and mechanosensory transduction.

The rapid progress in mechanobiology has brought together many scientific and engineering disciplines to work hand in hand toward better understanding of the role that mechanical force plays in functioning and evolution of different forms of life. New tools designed by engineers helped to develop new methods and techniques for investigation of mechanical properties of biological cells and tissues. This multidisciplinary approach made it clear that cell mechanics is tightly linked to intracellular signaling pathways, which directly regulate gene expression in response to mechanical stimuli originating outside or inside the cells. Mechanical stimuli act on mechanoreceptors which convert these stimuli into intracellular signals. In this chapter, we review the current knowledge about cell mechanics and the role cell mechanics plays for the function of mechanosensitive ion channels as a special class of mechanoreceptors functioning as molecular transducers of mechanical stimuli on a millisecond timescale.

A Tunable Microfluidic Device Enables Cargo Encapsulation by Cell- or Organelle-Sized Lipid Vesicles Comprising Asymmetric Lipid Bilayers.

Cellular membranes play host to a wide variety of morphologically and chemically complex processes. Although model membranes, like liposomes, are already widely used to reconstitute and study these processes, better tools are needed for making model bilayers that faithfully mimic cellular membranes. Existing methods for fabricating cell-sized (µm) or organelle-sized (tens to hundreds of nanometers) lipid vesicles have distinctly different requirements. Of particular note for biology, it remains challenging for any technique to efficiently encapsulate fragile cargo molecules or to generate liposomes with stable, asymmetric lipid leaflets within the bilayer. Here a tunable microfluidic device and protocol for fabricating liposomes with desired diameters ranging from ≈10 µm to ≈100 nm are described. Lipid vesicle size is templated by the simple inclusion of a polycarbonate filter within the microfluidic system and tuned with flow rate. It is shown that the vesicles made with this device are stable, unilamellar, lipid asymmetric, and capable of supporting transmembrane protein assembly, peripheral membrane protein binding, as well as soluble cargo encapsulation (including designer nanocages for biotechnology applications). These fabricated vesicles provide a new platform for studying the biophysically rich processes found within lipid-lipid and lipid-protein systems typically associated with cellular membranes.

FDM 3D Printing of High-Pressure, Heat-Resistant, Transparent Microfluidic Devices.

Transparent surfaces within microfluidic devices are essential for accurate quantification of chemical, biological, and mechanical interactions. Here, we report how to create low-cost, rapid 3D-printed microfluidic devices that are optically free from artifacts and have transparent surfaces suitable for visualizing a variety of fluid phenomenon. The methodology described here can be used for creating high-pressure microfluidic systems (significantly higher than PDMS-glass bonding). We develop methods for annealing Poly-Lactic Acid (PLA) microfluidic devices demonstrating heat resistance typically not achievable with other plastic materials. We show DNA melting and subsequent fluorescent imaging analysis, opening the door to other high-temperature applications. The FDM techniques demonstrated here allow for fabrication of microfluidic devices for precise visualization of interfacial dynamics, whether mixing between two laminar streams or droplet tracking. In addition to these characterizations, we include a printer troubleshooting guide and printing recipes for device fabrication to facilitate FDM printing for microfluidic device development.

The submerged printing of cells onto a modified surface using a continuous flow microspotter.

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

A critical comparison of protein microarray fabrication technologies.

Of the diverse analytical tools used in proteomics, protein microarrays possess the greatest potential for providing fundamental information on protein, ligand, analyte, receptor, and antibody affinity-based interactions, binding partners and high-throughput analysis. Microarrays have been used to develop tools for drug screening, disease diagnosis, biochemical pathway mapping, protein-protein interaction analysis, vaccine development, enzyme-substrate profiling, and immuno-profiling. While the promise of the technology is intriguing, it is yet to be realized. Many challenges remain to be addressed to allow these methods to meet technical and research expectations, provide reliable assay answers, and to reliably diversify their capabilities. Critical issues include: (1) inconsistent printed microspot morphologies and uniformities, (2) low signal-to-noise ratios due to factors such as complex surface capture protocols, contamination, and static or no-flow mass transport conditions, (3) inconsistent quantification of captured signal due to spot uniformity issues, (4) non-optimal protocol conditions such as pH, temperature, drying that promote variability in assay kinetics, and lastly (5) poor protein (e.g., antibody) printing, storage, or shelf-life compatibility with common microarray assay fabrication methods, directly related to microarray protocols. Conventional printing approaches, including contact (e.g., quill and solid pin), non-contact (e.g., piezo and inkjet), microfluidics-based, microstamping, lithography, and cell-free protein expression microarrays, have all been used with varying degrees of success with figures of merit often defined arbitrarily without comparisons to standards, or analytical or fiduciary controls. Many microarray performance reports use bench top analyte preparations lacking real-world relevance, akin to "fishing in a barrel", for proof of concept and determinations of figures of merit. This review critiques current protein-based microarray preparation techniques commonly used for analytical and function-based proteomics and their effects on array-based assay performance.

Continuous scaling 3d micro flow printing for improved spot morphology in protein microarrays - biomed 2013.

The protein microarray platform while innovative still poses a number of challenges which can only be met through creative and sophisticated system design. Pin printing while allowing for flexibility as to the type of medium printed does not offer the kind of spot reproducibility that a very sensitive application may require. The Continuous Flow Microspotter (CFM) was designed to not only allow for flexibility and reproducibility but to also achieve solution stability through flow scaling. This study uses the emerging CFM for printing protein and antibodies three dimensionally for general protein microarray applications. Consistent spot morphology, a continual and persistent problem in traditional pin printed microarrays, was compared under variable printed flow rates. The final assessment was performed using a rudimentary shear model. Force effects discussion and statistical data was used to demonstrate the versatility of the system.

Sensitivity of protein array deposition using continuous flow printing for fluorescent microarray applications - biomed 2013.

The promise of antibody and protein microarrays to revolutionize disease diagnostics has failed to live up to the hype primarily due to the problems associated with the printing of the antibodies and/or proteins onto the detection surface. The current standard in printing proteins is pin printing. An alternative to the pin printer is the continuous-flow microspotter (CFM), a protein printer that uses microfluidic flow to print down the proteins. The advantages of the CFM include consistent spot morphology, spot-to-spot uniformity and enhanced surface concentration. Further, the CFM is effective at capturing proteins and antibodies from either dilute or complex (e.g. blood or tissue) samples. In this study, the sensitivity of CFM printing Cy3 and Cy5 fluorescently labeled proteins was determined. Values were obtained at low concentrations tens of ng/mL with low coefficients of variation. Thus, the CFM can effectively print and quantify proteins and antibodies from low concentration and complex buffered samples.

Manual annotation of protein interactions.

Protein interactions are the basic building blocks for assembly of pathways and networks. Almost any biologically meaningful functionality (for instance, linear signaling pathways, chains of metabolic reactions, transcription factor dimmers, protein complexes of transcriptosome, gene-disease associations) can be represented as a combination of binary relationships between "network objects" (genes, proteins, RNA species, bioactive compounds). Naturally, the assembled pathways and networks are only as good as their "weakest" link (i.e., a wrongly assigned interaction), and the errors multiply in multi-step pathways. Therefore, the utility of "systems biology" is fundamentally dependent on quality and relevance of protein interactions. The second important parameter is the sheer number of interactions assembled in the database. One needs a "critical mass" of species-specific interactions in order to build cohesive networks for a gene list, not a constellation of non-connected proteins and protein pairs. The third issue is semantic consistency between interactions of different types. Transient physical signal transduction interactions, reactions of endogenous metabolism, transcription factor-promoter binding, and kinetic drug-target interactions are all very different in nature. Yet, they have to fit well into one database format and be consistent in order to be useful in reconstruction of cellular processes.High-quality protein interactions are available in peer-reviewed "small experiment" literature and, to a much smaller extent, patents. However, it is very challenging to find the interactions, annotate with searchable (and computable) parameters, catalogue in the database format in computer readable form, and assemble into a database. There are hundreds of thousands of mammalian interactions scattered in tens of thousands of papers in a few thousands of scientific journals. There are no widely used standards for reporting the interactions in scientific texts and, therefore, text-mining tools have only limited applicability. In order to generate a meaningful database of protein interactions, one needs a well-developed technology of manual curation, equipped with computational solutions, managerial procedures, quality control, and users' feedback. Here we describe our ever-evolving annotation approach, the important annotation issues and our solutions, and the mammalian protein interactions database MetaBase which we have been working on for over 8 years.