Rami Fishler

Particle dynamics and deposition in true-scale pulmonary acinar models.

Particle transport phenomena in the deep alveolated airways of the lungs (i.e. pulmonary acinus) govern deposition outcomes following inhalation of hazardous or pharmaceutical aerosols. Yet, there is still a dearth of experimental tools for resolving acinar particle dynamics and validating numerical simulations. Here, we present a true-scale experimental model of acinar structures consisting of bifurcating alveolated ducts that capture breathing-like wall motion and ensuing respiratory acinar flows. We study experimentally captured trajectories of inhaled polydispersed smoke particles (0.2 to 1 μm in diameter), demonstrating how intrinsic particle motion, i.e. gravity and diffusion, is crucial in determining dispersion and deposition of aerosols through a streamline crossing mechanism, a phenomenon paramount during flow reversal and locally within alveolar cavities. A simple conceptual framework is constructed for predicting the fate of inhaled particles near an alveolus by identifying capture and escape zones and considering how streamline crossing may shift particles between them. In addition, we examine the effect of particle size on detailed deposition patterns of monodispersed microspheres between 0.1–2 μm. Our experiments underline local modifications in the deposition patterns due to gravity for particles ≥0.5 μm compared to smaller particles, and show good agreement with corresponding numerical simulations.

Acinus-on-a-chip: A microfluidic platform for pulmonary acinar flows

Convective respiratory flows in the pulmonary acinus and their influence on the fate of inhaled particles are typically studied using computational fluid dynamics (CFD) or scaled-up experimental models. However, experiments that replicate several generations of the acinar tree while featuring cyclic wall motion have not yet been realized. Moreover, current experiments generally capture only flow dynamics, without inhaled particle dynamics, due to difficulties in simultaneously matching flow and particle dynamics. In an effort to overcome these limitations, we introduce a novel microfluidic device mimicking acinar flow characteristics directly at the alveolar scale. The model features an anatomically-inspired geometry that expands and contracts periodically with five dichotomously branching airway generations lined with alveolar-like cavities. We use micro-particle image velocimetry with a glycerol solution as the carrying fluid to quantitatively characterize detailed flow patterns within the device and reveal experimentally for the first time a gradual transition of alveolar flow patterns along the acinar tree from recirculating to radial streamlines, in support of hypothesized predictions from past CFD simulations. The current measurements show that our microfluidic system captures the underlying characteristics of the acinar flow environment, including Reynolds and Womersley numbers as well as cyclic wall displacements and alveolar flow patterns at a realistic length scale. With the use of air as the carrying fluid, our miniaturized platform is anticipated to capture both particle and flow dynamics and serve in the near future as a promising in vitro tool for investigating the mechanisms of particle deposition deep in the lung.

Mixed alkanethiol monolayers on submicrometric gold patterns: a controlled platform for studying cell-ligand interactions.

Nanoscale organization of surface ligands often has a critical effect on cell-surface interactions. We have developed an experimental system that allows a high degree of control over the 2-D spatial distribution of ligands. As a proof of concept, we used the developed system to study how T-cell activation is independently affected by antigen density and antigen amount per cell. Arrays of submicrometer gold islands at varying surface coverage were defined on silicon by electron beam lithography (EBL). The gold islands were functionalized with alkanethiol self-assembled monolayers (SAMs) containing a small antigen, 2,4,6-trinotrophenyl (TNP), at various densities. Genetically engineered T-cell hybridomas expressing TNP-specific chimeric T-cell antigen receptor (CAR) were cultured on the SAMs, and their activation was assessed by IL-2 secretion and CD69 expression. It was found that, at constant antigen density, activation increased monotonically with the amount of antigen, while at constant antigen amount activation was maximal at an intermediate antigen density, whose value was independent of the amount of antigen.

The role of respiratory flow asynchrony on convective mixing in the pulmonary acinus

Fine aerosol transport in the alveolated regions of the lungs is intrinsically coupled to alveolar flow patterns driven by lung breathing motions. Hence, understanding acinar flow characteristics is critical in determining local aerosol deposition sites. To date, inhaled aerosol dynamics have been mainly investigated using self-similar expanding lung models, although it is known that anisotropic lung motions exist and thus, potentially alter flow characteristics and enhance convective mixing. Using both experimental and numerical approaches, we assess here the influence of respiratory flow asynchrony on convective mixing by investigating alveolar flow patterns and massless particle transport for increasing phase lags between local wall motion and acinar ductal flows. Experimental results using a microfluidic platform, as well as numerical simulations, suggest that alveolar flow patterns are time-dependent in contrast to quasi-steady phenomena that pertain under synchronous conditions. To capture statistics of convective mixing, we numerically track massless tracers over multiple breathing cycles using anatomically inspired models of alveolated airways. By systemically probing various degrees of phase lag, our results underline the strong correlation between the magnitude of particle dispersion and flow asynchrony. In particular, we find that the dispersion of massless particles in acinar ducts is dramatically increased under flow asynchrony, relative to local, isolated alveolar cavity mixing. Despite the simplicity of the present models, our work highlights the critical role of respiratory flow asynchrony in governing the fate of fine inhaled particles in the pulmonary acinus.

Streamline crossing: An essential mechanism for aerosol dispersion in the pulmonary acinus

The dispersion of inhaled microparticles in the pulmonary acinus of the lungs is often attributed to the complex interplay between convective mixing, due to irreversible flows, and intrinsic particle motion (i.e. gravity and diffusion). However, the role of each mechanism, the exact nature of such interplay between them and their relative importance still remain unclear. To gain insight into these dispersive mechanisms, we track liquid-suspended microparticles and extract their effective diffusivities inside an anatomically-inspired microfluidic acinar model. Such results are then compared to experiments and numerical simulations in a straight channel. While alveoli of the proximal acinar generations exhibit convective mixing characteristics that lead to irreversible particle trajectories, this local effect is overshadowed by a more dominant dispersion mechanism across the ductal branching network that arises from small but significant streamline crossing due to intrinsic diffusional motion in the presence of high velocity gradients. We anticipate that for true airborne particles, which exhibit much higher intrinsic motion, streamline crossing would be even more significant.

Biomimetics of fetal alveolar flow phenomena using microfluidics

At the onset of life in utero, the respiratory system begins as a liquid-filled tubular organ and undergoes significant morphological changes during fetal development towards establishing a respiratory organ optimized for gas exchange. As airspace morphology evolves, respiratory alveolar flows have been hypothesized to exhibit evolving flow patterns. In the present study, we have investigated flow topologies during increasing phases of embryonic life within an anatomically inspired microfluidic device, reproducing real-scale features of fetal airways representative of three distinct phases of in utero gestation. Micro-particle image velocimetry measurements, supported by computational fluid dynamics simulations, reveal distinct respiratory alveolar flow patterns throughout different stages of fetal life. While attached, streamlined flows characterize the shallow structures of premature alveoli indicative of the onset of saccular stage, separated recirculating vortex flows become the signature of developed and extruded alveoli characteristic of the advanced stages of fetal development. To further mimic physiological aspects of the cellular environment of developing airways, our biomimetic devices integrate an alveolar epithelium using the A549 cell line, recreating a confluent monolayer that produces pulmonary surfactant. Overall, our in vitro biomimetic fetal airways model delivers a robust and reliable platform combining key features of alveolar morphology, flow patterns, and physiological aspects of fetal lungs developing in utero.

A Microfluidic Model of Biomimetically Breathing Pulmonary Acinar Airways.

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards optimizing drug inhalation techniques as well as predicting deposition patterns of potentially toxic airborne particles in the pulmonary alveoli. Here, soft-lithography techniques are used to fabricate complex acinar-like airway structures at the truthful anatomical length-scales that reproduce physiological acinar flow phenomena in an optically accessible system. The microfluidic device features 5 generations of bifurcating alveolated ducts with periodically expanding and contracting walls. Wall actuation is achieved by altering the pressure inside water-filled chambers surrounding the thin PDMS acinar channel walls both from the sides and the top of the device. In contrast to common multilayer microfluidic devices, where the stacking of several PDMS molds is required, a simple method is presented to fabricate the top chamber by embedding the barrel section of a syringe into the PDMS mold. This novel microfluidic setup delivers physiological breathing motions which in turn give rise to characteristic acinar air-flows. In the current study, micro particle image velocimetry (µPIV) with liquid suspended particles was used to quantify such air flows based on hydrodynamic similarity matching. The good agreement between µPIV results and expected acinar flow phenomena suggest that the microfluidic platform may serve in the near future as an attractive in vitro tool to investigate directly airborne representative particle transport and deposition in the acinar regions of the lungs.

Biomimetics of the pulmonary environment in vitro: A microfluidics perspective

The entire luminal surface of the lungs is populated with a complex yet confluent, uninterrupted airway epithelium in conjunction with an extracellular liquid lining layer that creates the air-liquid interface (ALI), a critical feature of healthy lungs. Motivated by lung disease modelling, cytotoxicity studies, and drug delivery assessments amongst other, in vitro setups have been traditionally conducted using macroscopic cultures of isolated airway cells under submerged conditions or instead using transwell inserts with permeable membranes to model the ALI architecture. Yet, such strategies continue to fall short of delivering a sufficiently realistic physiological in vitro airway environment that cohesively integrates at true-scale three essential pillars: morphological constraints (i.e., airway anatomy), physiological conditions (e.g., respiratory airflows), and biological functionality (e.g., cellular makeup). With the advent of microfluidic lung-on-chips, there have been tremendous efforts towards designing biomimetic airway models of the epithelial barrier, including the ALI, and leveraging such in vitro scaffolds as a gateway for pulmonary disease modelling and drug screening assays. Here, we review in vitro platforms mimicking the pulmonary environment and identify ongoing challenges in reconstituting accurate biological airway barriers that still widely prevent microfluidic systems from delivering mainstream assays for the end-user, as compared to macroscale in vitro cell cultures. We further discuss existing hurdles in scaling up current lung-on-chip designs, from single airway models to more physiologically realistic airway environments that are anticipated to deliver increasingly meaningful whole-organ functions, with an outlook on translational and precision medicine.

Particle sizing of pharmaceutical aerosols via direct imaging of particle settling velocities

Abstract We present a novel method for characterizing in near real‐time the aerodynamic particle size distributions from pharmaceutical inhalers. The proposed method is based on direct imaging of airborne particles followed by a particle‐by‐particle measurement of settling velocities using image analysis and particle tracking algorithms. Due to the simplicity of the principle of operation, this method has the potential of circumventing potential biases of current real‐time particle analyzers (e.g. Time of Flight analysis), while offering a cost effective solution. The simple device can also be constructed in laboratory settings from off‐the‐shelf materials for research purposes. To demonstrate the feasibility and robustness of the measurement technique, we have conducted benchmark experiments whereby aerodynamic particle size distributions are obtained from several commercially‐available dry powder inhalers (DPIs). Our measurements yield size distributions (i.e. MMAD and GSD) that are closely in line with those obtained from Time of Flight analysis and cascade impactors suggesting that our imaging‐based method may embody an attractive methodology for rapid inhaler testing and characterization. In a final step, we discuss some of the ongoing limitations of the current prototype and conceivable routes for improving the technique. Graphical abstract Figure. No caption available.

Microfluidic in Vitro Platforms of Pulmonary Alveolar Physiology

Recreating realistic features of the pulmonary acinus within an experimental model system is among the great challenges of modern respiratory physiology. Intricate anatomical architecture, distinct physiological flow patterns and complex cellular functions all render limited experimental approaches, capturing only some aspects of acinar airway physiology. Microfluidic-based in vitro devices (μFIVDs) offer attractive advantages over conventional in vitro models, and thus miniaturized technologies are becoming more frequently implemented to recreate biomimetic models of the pulmonary tract. However, current μFIVDs still lack critical physiological aspects of the pulmonary acinus; models are often limited to single channels and operate under submerged conditions that are loosely reflecting the realistic acinar environment. Here, we present an anatomically-inspired and physiologically-relevant cell-based in vitro microfluidic platform that combines a multi-generation design of ductal airways and alveolar spaces and integrates confluent monolayers of alveolar epithelium, recreating either fluid-submerged or air-exposed environments. Our microfluidic platform provides robust tools to study numerous aspects of pulmonary physiology, including varying alveolar morphology during fetal development, the propagation of liquid plugs alog airways and cytotoxicity of airborne particles deposited on alveolar walls. Overall, we propose a versatile model that captures anatomical and physiological pulmonary functionalities while preserving homeostatic cellular microenvironments.