A novel jamming phase diagram links tumor invasion to non-equilibrium phase separation.

It is well established that the early malignant tumor invades surrounding extracellular matrix (ECM) in a manner that depends upon material properties of constituent cells, surrounding ECM, and their interactions. Recent studies have established the capacity of the invading tumor spheroids to evolve into coexistent solid-like, fluid-like, and gas-like phases. Using breast cancer cell lines invading into engineered ECM, here we show that the spheroid interior develops spatial and temporal heterogeneities in material phase which, depending upon cell type and matrix density, ultimately result in a variety of phase separation patterns at the invasive front. Using a computational approach, we further show that these patterns are captured by a novel jamming phase diagram. We suggest that non-equilibrium phase separation based upon jamming and unjamming transitions may provide a unifying physical picture to describe cellular migratory dynamics within, and invasion from, a tumor.

Epithelial layer unjamming shifts energy metabolism toward glycolysis.

In development of an embryo, healing of a wound, or progression of a carcinoma, a requisite event is collective epithelial cellular migration. For example, cells at the advancing front of a wound edge tend to migrate collectively, elongate substantially, and exert tractions more forcefully compared with cells many ranks behind. With regards to energy metabolism, striking spatial gradients have recently been reported in the wounded epithelium, as well as in the tumor, but within the wounded cell layer little is known about the link between mechanical events and underlying energy metabolism. Using the advancing confluent monolayer of MDCKII cells as a model system, here we report at single cell resolution the evolving spatiotemporal fields of cell migration speeds, cell shapes, and traction forces measured simultaneously with fields of multiple indices of cellular energy metabolism. Compared with the epithelial layer that is unwounded, which is non-migratory, solid-like and jammed, the leading edge of the advancing cell layer is shown to become progressively more migratory, fluid-like, and unjammed. In doing so the cytoplasmic redox ratio becomes progressively smaller, the NADH lifetime becomes progressively shorter, and the mitochondrial membrane potential and glucose uptake become progressively larger. These observations indicate that a metabolic shift toward glycolysis accompanies collective cellular migration but show, further, that this shift occurs throughout the cell layer, even in regions where associated changes in cell shapes, traction forces, and migration velocities have yet to penetrate. In characterizing the wound healing process these morphological, mechanical, and metabolic observations, taken on a cell-by-cell basis, comprise the most comprehensive set of biophysical data yet reported. Together, these data suggest the novel hypothesis that the unjammed phase evolved to accommodate fluid-like migratory dynamics during episodes of tissue wound healing, development, and plasticity, but is more energetically expensive compared with the jammed phase, which evolved to maintain a solid-like non-migratory state that is more energetically economical.

Unjamming and collective migration in MCF10A breast cancer cell lines.

Each cell comprising an intact, healthy, confluent epithelial layer ordinarily remains sedentary, firmly adherent to and caged by its neighbors, and thus defines an elemental constituent of a solid-like cellular collective [1,2]. After malignant transformation, however, the cellular collective can become fluid-like and migratory, as evidenced by collective motions that arise in characteristic swirls, strands, ducts, sheets, or clusters [3,4]. To transition from a solid-like to a fluid-like phase and thereafter to migrate collectively, it has been recently argued that cells comprising the disordered but confluent epithelial collective can undergo changes of cell shape so as to overcome geometric constraints attributable to the newly discovered phenomenon of cell jamming and the associated unjamming transition (UJT) [1,2,5-9]. Relevance of the jamming concept to carcinoma cells lines of graded degrees of invasive potential has never been investigated, however. Using classical in vitro cultures of six breast cancer model systems, here we investigate structural and dynamical signatures of cell jamming, and the relationship between them [1,2,10,11]. In order of roughly increasing invasive potential as previously reported, model systems examined included MCF10A, MCF10A.Vector; MCF10A.14-3-3ζ; MCF10.ErbB2, MCF10AT; and MCF10CA1a [12-15]. Migratory speed depended on the particular cell line. Unsurprisingly, for example, the MCF10CA1a cell line exhibited much faster migratory speed relative to the others. But unexpectedly, across different cell lines higher speeds were associated with enhanced size of cooperative cell packs in a manner reminiscent of a peloton [9]. Nevertheless, within each of the cell lines evaluated, cell shape and shape variability from cell-to-cell conformed with predicted structural signatures of cell layer unjamming [1]. Moreover, both structure and migratory dynamics were compatible with previous theoretical descriptions of the cell jamming mechanism [2,10,11,16,17]. As such, these findings demonstrate the richness of the cell jamming mechanism, which is now seen to apply across these cancer cell lines but remains poorly understood.

Single-step assembly of asymmetric vesicles.

Asymmetric vesicles are membranes in which amphiphiles are asymmetrically distributed between each membrane leaflet. This asymmetry dictates chemical and physical properties of these vesicles, enabling their use as more realistic models of biological cell membranes, which also are asymmetric, and improves their potential for drug delivery and cosmetic applications. However, their fabrication is difficult as the self-assembly of amphiphiles always leads to symmetric vesicles. Here, we report the use of water-in-oil-in-oil-in-water triple emulsion drops to direct the assembly of the two leaflets to form asymmetric vesicles. Different compositions of amphiphiles are dissolved in each of the two oil shells of the triple emulsion; the amphiphiles diffuse to the interfaces and adsorb differentially at each of the two oil/water interfaces of the triple emulsion. These middle oil phases dewet from the innermost water cores of the triple emulsion drops, leading to the formation of membranes with degrees of asymmetry up to 70%. The triple emulsion drops are fabricated using capillary microfluidics, enabling production of highly monodisperse drops at rates as high as 300 Hz. Vesicles produced by this method can very efficiently encapsulate many different ingredients; this further enhances the utility of asymmetric vesicles as artificial cells, bioreactors and delivery vehicles.

Evolution on the Biophysical Fitness Landscape of an RNA Virus.

Viral evolutionary pathways are determined by the fitness landscape, which maps viral genotype to fitness. However, a quantitative description of the landscape and the evolutionary forces on it remain elusive. Here, we apply a biophysical fitness model based on capsid folding stability and antibody binding affinity to predict the evolutionary pathway of norovirus escaping a neutralizing antibody. The model is validated by experimental evolution in bulk culture and in a drop-based microfluidics that propagates millions of independent small viral subpopulations. We demonstrate that along the axis of binding affinity, selection for escape variants and drift due to random mutations have the same direction, an atypical case in evolution. However, along folding stability, selection and drift are opposing forces whose balance is tuned by viral population size. Our results demonstrate that predictable epistatic tradeoffs between molecular traits of viral proteins shape viral evolution.

Matrix Production and Sporulation in Bacillus subtilis Biofilms Localize to Propagating Wave Fronts.

Bacterial biofilms are surface-attached microbial communities encased in self-produced extracellular polymeric substances. Here we demonstrate that during the development of Bacillus subtilis biofilms, matrix production is localized to an annular front propagating at the periphery and sporulation to a second front at a fixed distance at the interior. We show that within these fronts, cells switch off matrix production and transition to sporulation after a set time delay of ∼100 min. Correlation analyses of fluctuations in fluorescence reporter activity reveal that the fronts emerge from a pair of gene-expression waves of matrix production and sporulation. The localized expression waves travel across cells that are immobilized in the biofilm matrix in contrast to active cell migration or horizontal colony spreading. Our results suggest that front propagation arises via a local developmental program occurring at the level of individual bacterial cells, likely driven by nutrient depletion and metabolic by-product accumulation. A single-length scale and timescale couples the spatiotemporal propagation of both fronts throughout development. As a result, gene expression patterns within the advancing fronts collapse to self-similar expression profiles. Our findings highlight the key role of the localized cellular developmental program associated with the propagating front in describing biofilm growth.

Microfluidic Fabrication of Pluronic Vesicles with Controlled Permeability.

Block copolymers with a low hydrophilic-to-lipophilic balance form membranes that are highly permeable to hydrophilic molecules. Polymersomes with this type of membrane enable the controllable release of molecules without membrane rupture. However, these polymersomes are difficult to assemble because of their low hydrophobicity. Here, we report a microfluidic approach to the production of these polymersomes using double-emulsion drops with ultrathin shells as templates. The small thickness of the middle oil phase enables the attraction of the hydrophobic blocks of the polymers adsorbed at each of the oil/water interfaces of the double emulsions; this results in the dewetting of the oil from the surface of the innermost water drops of the double emulsions and the ultimate formation of the polymersome. This approach to polymersome fabrication enables control of the vesicle size and results in the efficient encapsulation of hydrophilic ingredients that can be released through the polymer membrane without membrane rupture. We apply our approach to the fabrication of Pluronic L121 vesicles and characterize the permeability of their membranes. Furthermore, we show that membrane permeability can be tuned by blending different Pluronic polymers. Our work thus describes a route to producing Pluronic vesicles that are useful for the controlled release of hydrophilic ingredients.

Probing phenotypic growth in expanding Bacillus subtilis biofilms.

We develop an optical imaging technique for spatially and temporally tracking biofilm growth and the distribution of the main phenotypes of a Bacillus subtilis strain with a triple-fluorescent reporter for motility, matrix production, and sporulation. We develop a calibration procedure for determining the biofilm thickness from the transmission images, which is based on Beer-Lambert's law and involves cross-sectioning of biofilms. To obtain the phenotype distribution, we assume a linear relationship between the number of cells and their fluorescence and determine the best combination of calibration coefficients that matches the total number of cells for all three phenotypes and with the total number of cells from the transmission images. Based on this analysis, we resolve the composition of the biofilm in terms of motile, matrix-producing, sporulating cells and low-fluorescent materials which includes matrix and cells that are dead or have low fluorescent gene expression. We take advantage of the circular growth to make kymograph plots of all three phenotypes and the dominant phenotype in terms of radial distance and time. To visualize the nonlocal character of biofilm growth, we also make kymographs using the local colonization time. Our technique is suitable for real-time, noninvasive, quantitative studies of the growth and phenotype distribution of biofilms which are either exposed to different conditions such as biocides, nutrient depletion, dehydration, or waste accumulation.

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders.

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (10(3)-10(6)). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution.

Rapid Assembly of Heterogeneous 3D Cell Microenvironments in a Microgel Array.

Heterogeneous 3D cell microenvironment arrays are rapidly assembled by combining surface-wettability-guided assembly and microdroplet-array-based operations. This approach enables precise control over individual shapes, sizes, chemical concentrations, cell density, and 3D spatial distribution of multiple components. This technique provides a cost-effective solution to meet the increasing demand of stem cell research, tissue engineering, and drug screening.

Drying kinetics driven by the shape of the air/water interface in a capillary channel.

We look at the drying process in a simple glass channel with dominant capillary effects as is the case in microfluidics. We find drying kinetics commonly observed for confined geometry, namely a constant period followed by a falling rate period. From visualization of the air/water interface with high resolution, we observe that the drying rate decreases without a drying front progression although this is the usually accepted mechanism for confined geometries. We show with FEM that in our specific geometry the falling rate period is due to changes in the shape of the air-water interface at the free surface where most evaporation occurs. Our simulations show that the sensitivity of the drying rate to the shape of the first air-water interface from the sample free surface implies that slight changes of the wetting or pinning conditions can significantly modify the drying rate.

Rapid, targeted and culture-free viral infectivity assay in drop-based microfluidics.

A key viral property is infectivity, and its accurate measurement is crucial for the understanding of viral evolution, disease and treatment. Currently viral infectivity is measured using plaque assays, which involve prolonged culturing of host cells, and whose measurement is unable to differentiate between specific strains and is prone to low number fluctuation. We developed a rapid, targeted and culture-free infectivity assay using high-throughput drop-based microfluidics. Single infectious viruses are incubated in a large number of picoliter drops with host cells for one viral replication cycle followed by in-drop gene-specific amplification to detect infection events. Using murine noroviruses (MNV) as a model system, we measure their infectivity and determine the efficacy of a neutralizing antibody for different variants of MNV. Our results are comparable to traditional plaque-based assays and plaque reduction neutralization tests. However, the fast, low-cost, highly accurate genomic-based assay promises to be a superior method for drug screening and isolation of resistant viral strains. Moreover our technique can be adapted to measuring the infectivity of other pathogens, such as bacteria and fungi.

Artifact-Free Quantification and Sequencing of Rare Recombinant Viruses by Using Drop-Based Microfluidics.

Recombination is an important driver in the evolution of viruses and thus is key to understanding viral epidemics and improving strategies to prevent future outbreaks. Characterization of rare recombinant subpopulations remains technically challenging because of artifacts such as artificial recombinants, known as chimeras, and amplification bias. To overcome this, we have developed a high-throughput microfluidic technique with a second verification step in order to amplify and sequence single recombinant viruses with high fidelity in picoliter drops. We obtained the first artifact-free estimate of in vitro recombination rate between murine norovirus strains MNV-1 and WU20 co-infecting a cell (P(rec) = 3.3 × 10(-4) ± 2 × 10(-5) ) for a 1205 nt region. Our approach represents a time- and cost-effective improvement over current methods, and can be adapted for genomic studies requiring artifact- and bias-free selective amplification, such as microbial pathogens, or rare cancer cells.

Label-free single-cell protein quantification using a drop-based mix-and-read system.

Quantitative protein analysis of single cells is rarely achieved due to technical difficulties of detecting minute amounts of proteins present in one cell. We develop a mix-and-read assay for drop-based label-free protein analysis of single cells. This high-throughput method quantifies absolute, rather than relative, amounts of proteins and does not involve antibody labeling or mass spectrometry.

Microcapsules for Enhanced Cargo Retention and Diversity.

Prevention of undesired leakage of encapsulated materials prior to triggered release presents a technological challenge for the practical application of microcapsule technologies in agriculture, drug delivery, and cosmetics. A microfluidic approach is reported to fabricate perfluoropolyether (PFPE)-based microcapsules with a high core-shell ratio that show enhanced retention of encapsulated actives. For the PFPE capsules, less than 2% leakage of encapsulated model compounds, including Allura Red and CaCl2 , over a four week trial period is observed. In addition, PFPE capsules allow cargo diversity by the fabrication of capsules with either a water-in-oil emulsion or an organic solvent as core. Capsules with a toluene-based core begin a sustained release of hydrophobic model encapsulants immediately upon immersion in an organic continuous phase. The major contribution on the release kinetics stems from the toluene in the core. Furthermore, degradable silica particles are incorporated to confer porosity and functionality to the otherwise chemically inert PFPE-based polymer shell. These results demonstrate the capability of PFPE capsules with large core-shell ratios to retain diverse sets of cargo for extended periods and make them valuable for controlled release applications that require a low residual footprint of the shell material.

Locomotor benefits of being a slender and slick sand swimmer.

Squamates classified as 'subarenaceous' possess the ability to move long distances within dry sand; body elongation among sand and soil burrowers has been hypothesized to enhance subsurface performance. Using X-ray imaging, we performed the first kinematic investigation of the subsurface locomotion of the long, slender shovel-nosed snake (Chionactis occipitalis) and compared its biomechanics with those of the shorter, limbed sandfish lizard (Scincus scincus). The sandfish was previously shown to maximize swimming speed and minimize the mechanical cost of transport during burial. Our measurements revealed that the snake also swims through sand by propagating traveling waves down the body, head to tail. Unlike the sandfish, the snake nearly followed its own tracks, thus swimming in an approximate tube of self-fluidized granular media. We measured deviations from tube movement by introducing a parameter, the local slip angle, ßs, which measures the angle between the direction of movement of each segment and body orientation. The average ßs was smaller for the snake than for the sandfish; granular resistive force theory (RFT) revealed that the curvature utilized by each animal optimized its performance. The snake benefits from its slender body shape (and increased vertebral number), which allows propagation of a higher number of optimal curvature body undulations. The snake's low skin friction also increases performance. The agreement between experiment and RFT combined with the relatively simple properties of the granular 'frictional fluid' make subarenaceous swimming an attractive system to study functional morphology and bauplan evolution.

Eccentric rheometry for viscoelastic characterization of small, soft, anisotropic, and irregularly shaped biopolymer gels and tissue biopsies.

Quantification of the physical properties of tissue biopsies and cell-remodeled hydrogels is critical for understanding tissue development and pathophysiological tissue remodeling. However, due to the low modulus, small size, irregular shape, and anisotropy of samples from these materials, accurate viscoelastic characterization using standard rheometric methods is problematic. The goal of this work is to utilize image analysis to extend rotational rheometry to these samples. In this method, the sample is offset to increase the torque generated; a custom clear glass geometry, right angle prism, and camera are used to determine the exact shape and location of the sample relative to the axis of rotation for calculation of the sample shear modulus, G'. Values of G' for standard polydimethylsiloxane gels tested in centered and eccentric configurations were not statistically different (respectively 137 ± 37 kPa and 126 ± 8 kPa, p = 0.58), indicating accuracy of the method. Additionally, G' values from circular and irregularly shaped collagen gels yielded equivalent results (31 ± 1.8 Pa and 31 ± 5.1 Pa, p = 0.29). A blood clot and a lipid plaque sample recovered from human patients (G' ~ 4 kPa) were successfully tested with this method demonstrating applicability to clinical diagnostics.

Rheology of evolving bidisperse granular media.

We investigate the mixing of bidisperse distributions of spherical particles for slowly rotating vanes by pouring smaller beads, diameter d(1), onto a monodisperse bed composed of larger beads, diameter d(0), and monitoring the torque and lift forces. If the mixing beads are too small, d(1)/d(0)<0.05, the drag and lift are unaltered. Otherwise smaller beads displace larger beads from the shearing region, and if present in sufficient quantity both the drag and lift diminish to values of a monodisperse bed composed entirely of the smaller beads. We observe reductions in the torque up to 70%. The rate at which smaller beads leave the shearing region decreases as their size relative to the larger beads increases, and for d(1)/d(0)>0.155 the smaller beads remain inside the shearing region.

Rheology of draining steady-state foams.

Aqueous foams continuously age due to fluid drainage and bubble coarsening, which makes it difficult to perform steady-state rheological measurements. Consequently we have developed the foam drainage rheology technique, where perfusion counteracts fluid drainage and bubble replenishment counteracts bubble coarsening during measurement of the shear stresses by a rheometer. We evaluate published power-law and Herschel-Bulkley models and find that parameters derived from emulsion experiments cannot describe our results. We propose a hybrid model, which combines our earlier film-shearing model, where the film thickness depends on liquid volume fraction, with a Herschel-Bulkley shear-rate dependence.

Rheology of steady-state draining foams.

We developed the foam drainage rheology technique in order to perform rheological measurements of aqueous foams at a set liquid fraction epsilon and fixed bubble radius R without the usual difficulties associated with fluid drainage and bubble coarsening. The shear stress exhibits a power-law dependence on strain-rate, tau approximately gamma[over]n where n approximately 0.2. The stress exhibits an inverse dependence on liquid content, tau approximately (1+h'epsilon)(-1), where h'=theta(10) exhibits a diminishing logarithmic trend with gamma[over]. We propose a model based upon film shearing as the dominant source of viscous dissipation.