Preparation and Structural Evaluation of Epithelial Cell Monolayers in a Physiologically Sized Microfluidic Culture Device.

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). However, studies in microfluidic channels with dimensions physiologically relevant to the terminal bronchioles of the human lung currently face several challenges, especially due to difficulties in establishing appropriate cell culture conditions, including media flow rates, within a given culture environment. The presented protocol describes an image-based approach to evaluate the structure of NCI-H441 human lung epithelial cells cultured in an oxygen-impermeable microfluidic channel with dimensions physiologically relevant to the terminal bronchioles of the human lung. Using phalloidin-based filamentous-actin staining, the cytoskeletal structures of the cells are revealed by confocal laser scanning microscopy, allowing for the visualization of individual as well as layered cells. Subsequent quantification determines whether the cell culture conditions being employed are producing uniform monolayers suitable for further experimentation. The protocol describes cell culture and layer evaluation methods in microfluidic channels and traditional fixed-well environments. This includes channel construction, cell culture and requisite conditions, fixation, permeabilization and staining, confocal microscopic imaging, image processing, and data analysis.

Electric Cell-Substrate Impedance Sensing (ECIS) as a Platform for Evaluating Barrier-Function Susceptibility and Damage from Pulmonary Atelectrauma.

Biophysical insults that either reduce barrier function (COVID-19, smoke inhalation, aspiration, and inflammation) or increase mechanical stress (surfactant dysfunction) make the lung more susceptible to atelectrauma. We investigate the susceptibility and time-dependent disruption of barrier function associated with pulmonary atelectrauma of epithelial cells that occurs in acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). This in vitro study was performed using Electric Cell-substrate Impedance Sensing (ECIS) as a noninvasive evaluating technique for repetitive stress stimulus/response on monolayers of the human lung epithelial cell line NCI-H441. Atelectrauma was mimicked through recruitment/derecruitment (RD) of a semi-infinite air bubble to the fluid-occluded micro-channel. We show that a confluent monolayer with a high level of barrier function is nearly impervious to atelectrauma for hundreds of RD events. Nevertheless, barrier function is eventually diminished, and after a critical number of RD insults, the monolayer disintegrates exponentially. Confluent layers with lower initial barrier function are less resilient. These results indicate that the first line of defense from atelectrauma resides with intercellular binding. After disruption, the epithelial layer community protection is diminished and atelectrauma ensues. ECIS may provide a platform for identifying damaging stimuli, ventilation scenarios, or pharmaceuticals that can reduce susceptibility or enhance barrier-function recovery.

Microscale distribution and dynamic surface tension of pulmonary surfactant normalize the recruitment of asymmetric bifurcating airways.

We investigate the influence of bifurcation geometry, asymmetry of daughter airways, surfactant distribution, and physicochemical properties on the uniformity of airway recruitment of asymmetric bifurcating airways. To do so, we developed microfluidic idealized in vitro models of bifurcating airways, through which we can independently evaluate the impact of carina location and daughter airway width and length. We explore the uniformity of recruitment and its relationship to the dynamic surface tension of the lining fluid and relate this behavior to the hydraulic (PHyd) and capillary (PCap) pressure drops. These studies demonstrate the extraordinary importance of PCap in stabilizing reopening, even in highly asymmetric systems. The dynamic surface tension of pulmonary surfactant is integral to this stability because it modulates PCap in a velocity-dependent manner. Furthermore, the surfactant distribution at the propagating interface can have a very large influence on recruitment stability by focusing surfactant preferentially to specific daughter airways. This implies that modification of the surfactant distribution through novel modes of ventilation could be useful in inducing uniformly recruited lungs, aiding in gas exchange, and reducing ventilator-induced lung injury.NEW & NOTEWORTHY The dynamic surface tension of pulmonary surfactant is integral to the uniformity of asymmetric bifurcation airway recruitments because it modulates capillary pressure drop in a velocity-dependent manner. Also, the surfactant distribution at the propagating interface can have a very large influence on recruitment stability by focusing surfactant preferentially to specific daughter airways. This implies that modification of the surfactant distribution through novel modes of ventilation could be useful in inducing uniformly recruited lungs, reducing ventilator-induced lung injury.

Flow characteristics of urethral catheters of the same caliber vary between manufacturers.

BACKGROUND: Clean intermittent catheterization (CIC) is frequently prescribed for bladder dysfunction, either per urethra or via a continent catheterizable channel. Small catheters may be required for infants or continent channels. Success with CIC is highly dependent upon patient and family compliance. The urinary flow rate through the catheter is an important factor, which can decrease CIC time and improve quality of life. There is little objective information regarding flow rate through urinary catheters to guide catheter recommendation or prescription. Clinically, we noted that there was a difference in flow among catheter brands, and we questioned if catheters of the same-labeled diameter exhibit the same flow characteristics, which could have implications for catheter selection. METHODS: Twenty-one commercially available male pediatric urinary catheters from nine brands were tested (11 straight tip, 10 coude tip). Nine of the 21 tested catheters had a hydrophilic coating. All tested catheters shared a 10F outer diameter. For each, microscopic imaging and a precision caliper were used to measure the inner diameter and tip inlet area. A hydraulic system modified from ASTM standard testing specifications was used to simulate bladder catheterization. Measurement of each catheter was repeated five times using three different static hydraulic pressures (20, 40 and 50 cmH2O). Catheter flow rate and structural measurements were identified and the fastest and slowest of the catheters are presented in the table. The variable flow rates between brands were due to the differences in catheter structural characteristics such as the inner diameter (ID) and the tip inlet area to inner lumen area ratio (AR). The maximum variation of flow rate of all tested 10F catheters was 48%, ID varied up to 22%, from 1.71 to 2.11 mm or 5.13-6.33F. AR varied up to 166%. The table delineates the fastest and slowest rates at three measured pressures. The outer diameter labeled 10F on packaging was true to size. CONCLUSIONS: Based on packaging information, providers, and patients are unable to predict urinary flow through a catheter and thus use information regarding flow rate to guide catheter selection. This information cannot be calculated based on ideal flow calculations and could be listed on packaging to assist physicians and families in selecting the optimal urinary catheter for CIC.

The unusual symmetric reopening effect induced by pulmonary surfactant.

This study investigates the stability of a finger of air as it propagates into a liquid-filled model of a liquid-filled model of a pulmonary bifurcation. We seek to elucidate the stability characteristics of the reopening of daughter airways, an event that may be important to the treatment of acute lung disease. To do so, we investigated the symmetry of reopening under conditions of nearly constant surface tension with 1) purified H2O or 2) an anionic surfactant (sodium dodecyl sulfate). Dynamic surface tension was investigated using pulmonary surfactant (Infasurf) with and without the presence of albumin. Flow visualization was accomplished using a microparticle image velocimetry (µ-PIV)/shadowgraph system through which we measured 1) the propagation velocity of the finger of air that reopens each daughter branch, and 2) the instantaneous and averaged velocity field of liquid phase surrounding the tip of the propagating bubble. Only pulmonary surfactant demonstrated the ability of maintaining a nearly symmetric propagation in the daughter channels, which is likely to lead to homogeneous airway reopening. In contrast, when pulmonary surfactant was inactivated by albumin or when the system was held at a nearly constant surface tension, reopening occurred asymmetrically. Our analysis suggests that Infasurf's dynamic surface tension qualities are important to stabilize the removal of liquid obstructions. This demonstrates a new important function of pulmonary surfactant for airway reopening of a multibranched network.

Lagrangian transport properties of pulmonary interfacial flows.

Disease states characterized by airway fluid occlusion and pulmonary surfactant insufficiency, such as respiratory distress syndrome, have a high mortality rate. Understanding the mechanics of airway reopening, particularly involving surfactant transport, may provide an avenue to increase patient survival via optimized mechanical ventilation waveforms. We model the occluded airway as a liquid-filled rigid tube with the fluid phase displaced by a finger of air that propagates with both mean and sinusoidal velocity components. Finite-time Lyapunov exponent (FTLE) fields are employed to analyse the convective transport characteristics, taking note of Lagrangian coherent structures (LCSs) and their effects on transport. The Lagrangian perspective of these techniques reveals flow characteristics that are not readily apparent by observing Eulerian measures. These analysis techniques are applied to surfactant-free velocity fields determined computationally, with the boundary element method, and measured experimentally with micro particle image velocimetry (µ-PIV). We find that the LCS divides the fluid into two regimes, one advected upstream (into the thin residual film) and the other downstream ahead of the advancing bubble. At higher oscillatory frequencies particles originating immediately inside the LCS experience long residence times at the air-liquid interface, which may be conducive to surfactant transport. At high frequencies a well-mixed attractor region is identified; this volume of fluid cyclically travels along the interface and into the bulk fluid. The Lagrangian analysis is applied to velocity data measured with 0.01 mg ml(-1) of the clinical pulmonary surfactant Infasurf in the bulk fluid, demonstrating flow field modifications with respect to the surfactant-free system that were not visible in the Eulerian frame.

µ-PIV measurements of the ensemble flow fields surrounding a migrating semi-infinite bubble.

Microscale particle image velocimetry (µ-PIV) measurements of ensemble flow fields surrounding a steadily-migrating semi-infinite bubble through the novel adaptation of a computer controlled linear motor flow control system. The system was programmed to generate a square wave velocity input in order to produce accurate constant bubble propagation repeatedly and effectively through a fused glass capillary tube. We present a novel technique for re-positioning of the coordinate axis to the bubble tip frame of reference in each instantaneous field through the analysis of the sudden change of standard deviation of centerline velocity profiles across the bubble interface. Ensemble averages were then computed in this bubble tip frame of reference. Combined fluid systems of water/air, glycerol/air, and glycerol/Si-oil were used to investigate flows comparable to computational simulations described in Smith and Gaver (2008) and to past experimental observations of interfacial shape. Fluorescent particle images were also analyzed to measure the residual film thickness trailing behind the bubble. The flow fields and film thickness agree very well with the computational simulations as well as existing experimental and analytical results. Particle accumulation and migration associated with the flow patterns near the bubble tip after long experimental durations are discussed as potential sources of error in the experimental method.

Ets-1 proto-oncogene as a potential predictor for poor prognosis of lung adenocarcinoma.

The proto-oncogene Ets-1 is a transcription factor that is known to regulate certain matrix metalloproteinases and plasminogen activator, which have been associated with malignant behaviors in solid carcinomas. We hypothesized that Ets-1 expression is also associated with tumor progression and a worse prognosis in lung carcinoma patients. To clarify the role of the Ets-1 proto-oncogene, the expression of Ets-1 in non-small cell lung carcinomas using 156 paraffin-embedded specimens was determined in surgically resected tissue samples. Immunohistochemical staining showed Ets-1 expression in 82 cases of 156 carcinomas (53%): 36 of 52 (69%) squamous cell carcinomas, 41 of 96 (43%) adenocarcinomas, and 5 of 8 (63%) other carcinomas. In adenocarcinomas, a higher proportion of acinar type expressed Ets-1 compared to papillary or alveolar type (p < 0.05). The proportion of adenocarcinoma that expressed Ets-1 increased with poorer histologic differentiation of the adenocarcinoma (p < 0.05). Ets-1 positive adenocarcinomas had a larger mean size than Ets-1 negative adenocarcinomas (p < 0.01). In adenocarcinoma patients, expression of Ets-1 was associated with disease-free (p = 0.09) and overall survivals (p < 0.05) after lung resection. Such relationship was not observed among squamous cell carcinoma patients. Our findings indicate that Ets-1 expression is related to histopathological differentiation, morphogenesis, and tumor progression of lung adenocarcinomas. Ets-1 appears to be a useful predictor of poor prognosis after surgical resection in lung adenocarcinoma patients. Ets-1 expression could be used to evaluate the malignant behaviors of lung adenocarcinomas.

Distal intramural spread is an independent prognostic factor for distant metastasis and poor outcome in patients with rectal cancer: a multivariate analysis.

BACKGROUND: The aim of this study was to clarify the prognostic value of distal intramural spread of tumor for survival and recurrence in patients with rectal cancer. METHODS: Microscopic distal intramural spread was examined in 134 consecutive specimens of resected rectal cancer. Correlations among distal intramural spread, established clinicopathologic factors, and patients' prognoses were examined by univariate and multivariate analyses. American Joint Committee on Cancer classification and stage groupings were used for tumor assessment. RESULTS: Thirty-three patients (24.6%) had distal intramural spread. Multivariate logistical regression analysis revealed that T3/T4 and M1 were independent predictive variables for the presence of distal intramural spread. Patients with distal intramural spread had a shorter disease-specific or disease-free survival time after curative surgery than those without distal intramural spread (P =.0003 and P =.0006, respectively). Most patients with distal intramural spread developed distant recurrence. Cox's regression with multiple covariates showed that distal intramural spread is an independent factor in predicting distant recurrence and worse outcomes after curative surgery in patients with rectal cancer. CONCLUSIONS: Distal intramural spread is an independent risk factor for distant metastasis and poor prognosis in patients with rectal cancer.