Measuring colloid-surface interaction forces in parallel using fluorescence centrifuge force microscopy.

Interactions between colloidal-scale structures govern the physical properties of soft and biological materials, and knowledge of the forces associated with these interactions is critical for understanding and controlling these materials. A common approach to quantify colloidal interactions is to measure the interaction forces between colloids and a fixed surface. The centrifuge force microscope (CFM), a miniaturized microscope inside a centrifuge, is capable of performing hundreds of force measurements in parallel over a wide force range (10-2 to 104 pN), but CFM instruments are not widely used to measure colloid-surface interaction forces. In addition, current CFM instruments rely on brightfield illumination and are not capable of fluorescence microscopy. Here we present a fluorescence CFM (F-CFM) that combines both fluorescence and brightfield microscopy and demonstrate its use for measuring microscale colloidal-surface interaction forces. The F-CFM operates at speeds up to 5000 RPM, 2.5× faster than those previously reported, yielding a 6.25× greater maximum force than previous instruments. A battery-powered GoPro video camera enables real-time viewing of the microscopy video on a mobile device, and frequency analysis of the audio signal correlates centrifuge rotational speed with the video signal. To demonstrate the capability of the F-CFM, we measure the force required to detach hundreds of electrostatically stabilized colloidal microspheres attached to a charged glass surface as a function of ionic strength and compare the resulting force distributions with an approximated DLVO theory. The F-CFM will enable microscale force measurements to be correlated with fluorescence imaging in soft and biological systems.

Evaluation of the Antimicrobial Efficacy of N-Acetyl-l-Cysteine, Rhamnolipids, and Usnic Acid-Novel Approaches to Fight Food-Borne Pathogens.

In the food industry, the increasing antimicrobial resistance of food-borne pathogens to conventional sanitizers poses the risk of food contamination and a decrease in product quality and safety. Therefore, we explored alternative antimicrobials N-Acetyl-l-cysteine (NAC), rhamnolipids (RLs), and usnic acid (UA) as a novel approach to prevent biofilm formation and reduce existing biofilms formed by important food-borne pathogens (three strains of Salmonella enterica and two strains of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus). Their effectiveness was evaluated by determining minimum inhibitory concentrations needed for inhibition of bacterial growth, biofilm formation, metabolic activity, and biofilm reduction. Transmission electron microscopy and confocal scanning laser microscopy followed by image analysis were used to visualize and quantify the impact of tested substances on both planktonic and biofilm-associated cells. The in vitro cytotoxicity of the substances was determined as a half-maximal inhibitory concentration in five different cell lines. The results indicate relatively low cytotoxic effects of NAC in comparison to RLs and UA. In addition, NAC inhibited bacterial growth for all strains, while RLs showed overall lower inhibition and UA inhibited only the growth of Gram-positive bacteria. Even though tested substances did not remove the biofilms, NAC represents a promising tool in biofilm prevention.

Live imaging analysis of human gastric epithelial spheroids reveals spontaneous rupture, rotation and fusion events.

Three-dimensional cultures of primary epithelial cells including organoids, enteroids and epithelial spheroids have become increasingly popular for studies of gastrointestinal development, mucosal immunology and epithelial infection. However, little is known about the behavior of these complex cultures in their three-dimensional culture matrix. Therefore, we performed extended time-lapse imaging analysis (up to 4 days) of human gastric epithelial spheroids generated from adult tissue samples in order to visualize the dynamics of the spheroids in detail. Human gastric epithelial spheroids cultured in our laboratory grew to an average diameter of 443.9 ± 34.6 µm after 12 days, with the largest spheroids reaching diameters of >1000 µm. Live imaging analysis revealed that spheroid growth was associated with cyclic rupture of the epithelial shell at a frequency of 0.32 ± 0.1/day, which led to the release of luminal contents. Spheroid rupture usually resulted in an initial collapse, followed by spontaneous re-formation of the spheres. Moreover, spheroids frequently rotated around their axes within the Matrigel matrix, possibly propelled by basolateral pseudopodia-like formations of the epithelial cells. Interestingly, adjacent spheroids occasionally underwent luminal fusion, as visualized by injection of individual spheroids with FITC-Dextran (4 kDa). In summary, our analysis revealed unexpected dynamics in human gastric spheroids that challenge our current view of cultured epithelia as static entities and that may need to be considered when performing spheroid infection experiments.

NMR Relaxometry to Characterize the Drug Structural Phase in a Porous Construct.

Nuclear magnetic resonance (NMR) frequency spectra and T2 relaxation time measurements, using a high-power radio frequency probe, are shown to characterize the presence of an amorphous drug in a porous silica construct. The results indicate the ability of non-solid-state NMR methods to characterize crystalline and amorphous solid structural phases in drugs. Two-dimensional T1- T2 magnetic relaxation time correlation experiments are shown to monitor the impact of relative humidity on the drug in a porous silica tablet.

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.

Liquid transport facilitated by channels in Bacillus subtilis biofilms.

Many bacteria on earth exist in surface-attached communities known as biofilms. These films are responsible for manifold problems, including hospital-acquired infections and biofouling, but they can also be beneficial. Biofilm growth depends on the transport of nutrients and waste, for which diffusion is thought to be the main source of transport. However, diffusion is ineffective for transport over large distances and thus should limit growth. Nevertheless, biofilms can grow to be very large. Here we report the presence of a remarkable network of well-defined channels that form in wild-type Bacillus subtilis biofilms and provide a system for enhanced transport. We observe that these channels have high permeability to liquid flow and facilitate the transport of liquid through the biofilm. In addition, we find that spatial variations in evaporative flux from the surface of these biofilms provide a driving force for the flow of liquid in the channels. These channels offer a remarkably simple system for liquid transport, and their discovery provides insight into the physiology and growth of biofilms.

Monodisperse Emulsion Drop Microenvironments for Bacterial Biofilm Growth.

In this work, microfluidic technology is used to rapidly create hundreds of thousands of monodisperse double and triple emulsion drops that serve as 3D microenvironments for the containment and growth of bacterial biofilms. The size of these drops, with diameters from tens to hundreds of micrometers, makes them amenable to rapid manipulation and analysis. This is demonstrated by using microscopy to visualize cellular differentiation of Bacillus subtilis biofilm communities within each drop and the bacterial biofilm microstructure. Biofilm growth is explored upon specific interfaces in double and triple emulsions and upon negative and positive radii of curvature. Biofilm attachment of matrix and flagella mutants is studied as well as biofilms of Pseudomonas aeruginosa. This is the first demonstration of biofilms grown in microscale emulsion drops, which serve as both templates and containers for biofilm growth and attachment. These microenvironments have the potential to transform existing high-throughput screening methods for bacterial biofilms.

Osmotic spreading of Bacillus subtilis biofilms driven by an extracellular matrix.

Bacterial biofilms are organized communities of cells living in association with surfaces. The hallmark of biofilm formation is the secretion of a polymeric matrix rich in sugars and proteins in the extracellular space. In Bacillus subtilis, secretion of the exopolysaccharide (EPS) component of the extracellular matrix is genetically coupled to the inhibition of flagella-mediated motility. The onset of this switch results in slow expansion of the biofilm on a substrate. Different strains have radically different capabilities in surface colonization: Flagella-null strains spread at the same rate as wild type, while both are dramatically faster than EPS mutants. Multiple functions have been attributed to the EPS, but none of these provides a physical mechanism for generating spreading. We propose that the secretion of EPS drives surface motility by generating osmotic pressure gradients in the extracellular space. A simple mathematical model based on the physics of polymer solutions shows quantitative agreement with experimental measurements of biofilm growth, thickening, and spreading. We discuss the implications of this osmotically driven type of surface motility for nutrient uptake that may elucidate the reduced fitness of the matrix-deficient mutant strains.

The jamming elasticity of emulsions stabilized by ionic surfactants.

Oil-in-water emulsions composed of colloidal-scale droplets are often stabilized using ionic surfactants that provide a repulsive interaction between neighboring droplet interfaces, thereby inhibiting coalescence. If the droplet volume fraction is raised rapidly by applying an osmotic pressure, the droplets remain disordered, undergo an ergodic-nonergodic transition, and jam. If the applied osmotic pressure approaches the Laplace pressure of the droplets, then the jammed droplets also deform. Because solid friction and entanglements cannot play a role, as they might with solid particulate or microgel dispersions, the shear mechanical response of monodisperse emulsions can provide critical insight into the interplay of entropic, electrostatic, and interfacial forces. Here, we introduce a model that can be used to predict the plateau storage modulus and yield stress of a uniform charge-stabilized emulsion accurately if the droplet radius, interfacial tension, surface potential, Debye screening length, and droplet volume fraction are known.

Antiviral responses in a Jamaican fruit bat intestinal organoid model of SARS-CoV-2 infection.

Bats are natural reservoirs for several zoonotic viruses, potentially due to an enhanced capacity to control viral infection. However, the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB, Artibeus jamaicensis) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Upon infection with SARS-CoV-2, increased viral RNA and subgenomic RNA was detected, but no infectious virus was released, indicating that JFB organoids support only limited viral replication but not viral reproduction. SARS-CoV-2 replication was associated with significantly increased gene expression of type I interferons and inflammatory cytokines. Interestingly, SARS-CoV-2 also caused enhanced formation and growth of JFB organoids. Proteomics revealed an increase in inflammatory signaling, cell turnover, cell repair, and SARS-CoV-2 infection pathways. Collectively, our findings suggest that primary JFB intestinal epithelial cells mount successful antiviral interferon responses and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.

Characterizing concentrated, multiply scattering, and actively driven fluorescent systems with confocal differential dynamic microscopy.

We introduce confocal differential dynamic microscopy (ConDDM), a new technique yielding information comparable to that given by light scattering but in dense, opaque, fluorescent samples of micron-sized objects that cannot be probed easily with other existing techniques. We measure the correct wave vector q-dependent structure and hydrodynamic factors of concentrated hard-sphere-like colloids. We characterize concentrated swimming bacteria, observing ballistic motion in the bulk and a new compressed-exponential scaling of dynamics, and determine the velocity distribution; by contrast, near the coverslip, dynamics scale differently, suggesting that bacterial motion near surfaces fundamentally differs from that of freely swimming organisms.

Granular Matrigel: restructuring a trusted extracellular matrix material for improved permeability.

Matrigel is a polymeric extracellular matrix material produced by mouse cancer cells. Over the past four decades, Matrigel has been shown to support a wide variety of two- and three-dimensional cell and tissue culture applications including organoids. Despite widespread use, transport of molecules, cells, and colloidal particles through Matrigel can be limited. These limitations restrict cell growth, viability, and function and limit Matrigel applications. A strategy to improve transport through a hydrogel without modifying the chemistry or composition of the gel is to physically restructure the material into microscopic microgels and then pack them together to form a porous material. These 'granular' hydrogels have been created using a variety of synthetic hydrogels, but granular hydrogels composed of Matrigel have not yet been reported. Here we present a drop-based microfluidics approach for structuring Matrigel into a three-dimensional, mesoporous material composed of packed Matrigel microgels, which we call granular Matrigel. We show that restructuring Matrigel in this manner enhances the transport of colloidal particles and human dendritic cells (DCs) through the gel while providing sufficient mechanical support for culture of human gastric organoids (HGOs) and co-culture of human DCs with HGOs.

Metabolomic Profiling and Mechanotransduction of Single Chondrocytes Encapsulated in Alginate Microgels.

Articular cartilage is comprised of two main components, the extracellular matrix (ECM) and the pericellular matrix (PCM). The PCM helps to protect chondrocytes in the cartilage from mechanical loads, but in patients with osteoarthritis, the PCM is weakened, resulting in increased chondrocyte stress. As chondrocytes are responsible for matrix synthesis and maintenance, it is important to understand how mechanical loads affect the cellular responses of chondrocytes. Many studies have examined chondrocyte responses to in vitro mechanical loading by embedding chondrocytes in 3-D hydrogels. However, these experiments are mostly performed in the absence of PCM, which may obscure important responses to mechanotransduction. Here, drop-based microfluidics is used to culture single chondrocytes in alginate microgels for cell-directed PCM synthesis that closely mimics the in vivo microenvironment. Chondrocytes formed PCM over 10 days in these single-cell 3-D microenvironments. Mechanotransduction studies were performed, in which single-cell microgels mimicking the cartilage PCM were embedded in high-stiffness agarose. After physiological dynamic compression in a custom-built bioreactor, microgels exhibited distinct metabolomic profiles from both uncompressed and monolayer controls. These results demonstrate the potential of single cell encapsulation in alginate microgels to advance cartilage tissue engineering and basic chondrocyte mechanobiology.

Chickensplash! Exploring the health concerns of washing raw chicken.

The Food and Drug Administration recommends against washing raw chicken due to the risk of transferring dangerous food-borne pathogens through splashed drops of water. Many cooks continue to wash raw chicken despite this warning, however, and there is a lack of scientific research assessing the extent of microbial transmission in splashed droplets. Here, we use large agar plates to confirm that bacteria can be transferred from the surface of raw chicken through splashing. We also identify and create a phylogenetic tree of the bacteria present on the chicken and the bacteria transferred during splashing. While no food-borne pathogens were identified, we note that organisms in the same genera as pathogens were transferred from the chicken surface through these droplets. Additionally, we show that faucet height, flow type, and surface stiffness play a role in splash height and distance. Using high-speed imaging to explore splashing causes, we find that increasing faucet height leads to a flow instability that can increase splashing. Furthermore, splashing from soft materials such as chicken can create a divot in the surface, leading to splashing under flow conditions that would not splash on a curved, hard surface. Thus, we conclude that washing raw chicken does risk pathogen transfer and cross-contamination through droplet ejection, and that changing washing conditions can increase or decrease the risk of splashing.

Shear-induced disruption of dense nanoemulsion gels.

The structural evolution and rheology of dense nanoemulsion gels, which have been formed by creating strong attractions between slippery nanodroplets, are explored as a function of steady shear rate using rheological small-angle neutron scattering (rheo-SANS). For applied stresses above the yield stress of the gel, the network yields, fracturing into aggregates that break and reform as they tumble and interact in the shear flow. The average aggregate size decreases with increasing shear rate; meanwhile, droplet rearrangements within the clusters, allowed by the slippery nature of the attractive interaction, increase the local density within the aggregates. At the highest shear rates, all clusters disaggregate completely into individual droplets.

Coupling fluid flow to hydrogel fluidic devices with reversible "pop-it" connections.

Hydrogels are soft, water-based polymer gels that are increasingly used to fabricate free-standing fluidic devices for tissue and biological engineering applications. For many of these applications, pressurized liquid must be driven through the hydrogel device. To couple pressurized liquid to a hydrogel device, a common approach is to insert tubing into a hole in the gel; however, this usually results in leakage and expulsion of the tubing, and other options for coupling pressurized liquid to hydrogels remain limited. Here, we describe a simple coupling approach where microfluidic tubing is inserted into a plastic, 3D-printed bulb-shaped connector, which "pops" into a 3D-printed socket in the gel. By systematically varying the dimensions of the connector relative to those of the socket entrance, we find an optimal head-socket ratio that provides maximum resistance to leakage and expulsion. The resulting connection can withstand liquid pressures on the order of several kilopascals, three orders of magnitude greater than traditional, connector-free approaches. We also show that two-sided connectors can be used to link multiple hydrogels to one another to build complex, reconfigurable hydrogel systems from modular components. We demonstrate the potential usefulness of these connectors by established long-term nutrient flow through a 3D-printed hydrogel device containing bacteria. The simple coupling approach outlined here will enable a variety of applications in hydrogel fluidics.

Symmetry breaking propulsion of magnetic microspheres in nonlinearly viscoelastic fluids.

Microscale propulsion impacts a diverse array of fields ranging from biology and ecology to health applications, such as infection, fertility, drug delivery, and microsurgery. However, propulsion in such viscous drag-dominated fluid environments is highly constrained, with time-reversal and geometric symmetries ruling out entire classes of propulsion. Here, we report the spontaneous symmetry-breaking propulsion of rotating spherical microparticles within non-Newtonian fluids. While symmetry analysis suggests that propulsion is not possible along the fore-aft directions, we demonstrate the existence of two equal and opposite propulsion states along the sphere's rotation axis. We propose and experimentally corroborate a propulsion mechanism for these spherical microparticles, the simplest microswimmers to date, arising from nonlinear viscoelastic effects in rotating flows similar to the rod-climbing effect. Similar possibilities of spontaneous symmetry-breaking could be used to circumvent other restrictions on propulsion, revising notions of microrobotic design and control, drug delivery, microscale pumping, and locomotion of microorganisms.

A Synthetic Hydrogel, VitroGel® ORGANOID-3, Improves Immune Cell-Epithelial Interactions in a Tissue Chip Co-Culture Model of Human Gastric Organoids and Dendritic Cells.

Immunosurveillance of the gastrointestinal epithelium by mononuclear phagocytes (MNPs) is essential for maintaining gut health. However, studying the complex interplay between the human gastrointestinal epithelium and MNPs such as dendritic cells (DCs) is difficult, since traditional cell culture systems lack complexity, and animal models may not adequately represent human tissues. Microphysiological systems, or tissue chips, are an attractive alternative for these investigations, because they model functional features of specific tissues or organs using microscale culture platforms that recreate physiological tissue microenvironments. However, successful integration of multiple of tissue types on a tissue chip platform to reproduce physiological cell-cell interactions remains a challenge. We previously developed a tissue chip system, the gut organoid flow chip (GOFlowChip), for long term culture of 3-D pluripotent stem cell-derived human intestinal organoids. Here, we optimized the GOFlowChip platform to build a complex microphysiological immune-cell-epithelial cell co-culture model in order to study DC-epithelial interactions in human stomach. We first tested different tubing materials and chip configurations to optimize DC loading onto the GOFlowChip and demonstrated that DC culture on the GOFlowChip for up to 20 h did not impact DC activation status or viability. However, Transwell chemotaxis assays and live confocal imaging revealed that Matrigel, the extracellular matrix (ECM) material commonly used for organoid culture, prevented DC migration towards the organoids and the establishment of direct MNP-epithelial contacts. Therefore, we next evaluated DC chemotaxis through alternative ECM materials including Matrigel-collagen mixtures and synthetic hydrogels. A polysaccharide-based synthetic hydrogel, VitroGel®-ORGANOID-3 (V-ORG-3), enabled significantly increased DC chemotaxis through the matrix, supported organoid survival and growth, and did not significantly alter DC activation or viability. On the GOFlowChip, DCs that were flowed into the chip migrated rapidly through the V-ORG matrix and reached organoids embedded deep within the chip, with increased interactions between DCs and gastric organoids. The successful integration of DCs and V-ORG-3 embedded gastric organoids into the GOFlowChip platform now permits real-time imaging of MNP-epithelial interactions and other investigations of the complex interplay between gastrointestinal MNPs and epithelial cells in their response to pathogens, candidate drugs and mucosal vaccines.

Long-term flow through human intestinal organoids with the gut organoid flow chip (GOFlowChip).

Human intestinal organoids (HIOs) are millimeter-scale models of the human intestinal epithelium and hold tremendous potential for advancing fundamental and applied biomedical research. HIOs resemble the native gut in that they consist of a fluid-filled lumen surrounded by a polarized epithelium and associated mesenchyme; however, their topologically-closed, spherical shape prevents flow through the interior luminal space, making the system less physiological and leading to the buildup of cellular and metabolic waste. These factors ultimately limit experimentation inside the HIOs. Here, we present a millifluidic device called the gut organoid flow chip (GOFlowChip), which we use to "port" HIOs and establish steady-state liquid flow through the lumen for multiple days. This long-term flow is enabled by the use of laser-cut silicone gaskets, which allow liquid in the device to be slightly pressurized, suppressing bubble formation. To demonstrate the utility of the device, we establish separate luminal and extraluminal flow and use luminal flow to remove accumulated waste. This represents the first demonstration of established liquid flow through the luminal space of a gastrointestinal organoid over physiologically relevant time scales. Flow cytometry results reveal that HIO cell viability is unaffected by long-term porting and luminal flow. We expect the real-time, long-term control over luminal and extraluminal contents provided by the GOFlowChip will enable a wide variety of studies including intestinal secretion, absorption, transport, and co-culture with intestinal microorganisms.

A Novel Gastric Spheroid Co-culture Model Reveals Chemokine-Dependent Recruitment of Human Dendritic Cells to the Gastric Epithelium.

BACKGROUND & AIMS: Gastric dendritic cells (DCs) control the adaptive response to infection with Helicobacter pylori, a major risk factor for peptic ulcer disease and gastric cancer. We hypothesize that DC interactions with the gastric epithelium position gastric DCs for uptake of luminal H pylori and promote DC responses to epithelial-derived mediators. The aim of this study was to determine whether the gastric epithelium actively recruits DCs using a novel co-culture model of human gastric epithelial spheroids and monocyte-derived DCs. METHODS: Spheroid cultures of primary gastric epithelial cells were infected with H pylori by microinjection. Co-cultures were established by adding human monocyte-derived DCs to the spheroid cultures and were analyzed for DC recruitment and antigen uptake by confocal microscopy. Protein array, gene expression polymerase chain reaction array, and chemotaxis assays were used to identify epithelial-derived chemotactic factors that attract DCs. Data from the co-culture model were confirmed using human gastric tissue samples. RESULTS: Human monocyte-derived DCs co-cultured with gastric spheroids spontaneously migrated to the gastric epithelium, established tight interactions with the epithelial cells, and phagocytosed luminally applied H pylori. DC recruitment was increased upon H pylori infection of the spheroids and involved the activity of multiple chemokines including CXCL1, CXCL16, CXCL17, and CCL20. Enhanced chemokine expression and DC recruitment to the gastric epithelium also was observed in H pylori-infected human gastric tissue samples. CONCLUSIONS: Our results indicate that the gastric epithelium actively recruits DCs for immunosurveillance and pathogen sampling through chemokine-dependent mechanisms, with increased recruitment upon active H pylori infection.