Microvascular Mimetics for the Study of Leukocyte-Endothelial Interactions

ByAlec

Here are the figures for Tejas’ paper that is in the final stage of drafting:

Figure 1. Microvessel Mimetic (µSiM-MVM) platform: design and assembly. (A) 2D schematic showing apical (luminal) and basal (abluminal) compartments separated by a silicon nanomembrane, which also acts as a scaffold for incorporating type 1 collagen gel as an extracellular matrix (ECM) mimic. Top and bottom channels are accessible using independent inlet/outlet ports. Indium tin oxide (ITO) electrodes are assembled to enable electrical resistance measurement. (B) Exploded 3D view of the microfluidic system illustrating the silicone gaskets, pressure sensitive adhesive (PSA) and electrodes. (C) Assembled device without electrodes (for visual clarity) highlighting the miniaturized nature and the transparency of the platform.
Figure 2. TEER and EC morphology under shear: (A) TEER measurement was performed over 48-52 hours post cell seeding in the µSiM-MVM. TEER rises during first 24 hours (red curve), after which it remains stable in the absence of shear (blue curve) or progresses to an elevated level in the presence of shear (brown curve). The addition of thrombin immediately causes the TEER to decay below 50% of its maximum value in less than half hour (black dashed line). Error bars represent standard error of mean with at least n=5. (B) Endothelial cells grown under static condition for 24 hours demonstrate cobblestone morphology as observed in the phase image on the top left, and the corresponding image analysis in MATLAB reveals the isotropic orientation of the cell population. Endothelial cells exposed to shear stress of 10 dynes cm-2 for 24 hours exhibit uniformly aligned morphology with a majority of the cells parallel to the flow direction (Y axis).
Figure 3. Neutrophil transmigration through a collagen matrix: (A) Even in the presence of endothelial cells and supplemented collagen I gel, neutrophils transmigrate to the abluminal channel. The presence of basal gel (below the nanomembrane) doesn’t significantly reduce the amount of migrated cell population as observed from the representative images of cells on the cover-slip 3 hours after addition (17.54 ± 4.29 % in absence of basal gel and, 19.89 ± 5.52% in the presence of basal gel, n=3 each; percentage of cells calculated by dividing the number of cells in the basal chamber 3 hours later to the number of cells in the top chamber at the onset of migration). In the absence of endothelial cells, neutrophils (hPMN) migrate fastest due to potential absence of any steric inhibition. Scale bar = 100 μm (B) Scanning electron microscopy (SEM) analysis shows the ability of hPMNs to transmigrate through the collagen gel, eventually residing on the underlying nanomembrane.
Figure 4. Optical clarity of μSiM devices permit real-time observations of EC junctions: (A) Phase time lapse images of neutrophils (yellow arrow) transmigrating through a HUVEC monolayer reveal potential separations in EC junctions (blue arrow). Scale bar = 10 μm (B) Live immunolabeling of HUVEC junctional proteins (VE-Cadherin; green) further highlights the disruption of the EC barrier during a neutrophil transmigration event. Scale bar = 20 μm
Figure 5. Neutrophils migrate uninhibited in the interstitial space in the absence of β1 integrin blocking (A) but remain trapped in the subendothelial space in the presence of the blocking antibody against β1 integrins (B). Percentage of migration from the apical to basal compartments under the influence of basally added fMLP over the duration of 3 hours reduces significantly in response to β1 integrin blocking (C). Error bars represent standard error of mean; n=3 for each condition. (D) The disruption of 3D migration of β1 integrin-blocked neutrophils was again revealed under SEM. The yellow inset shows the zoomed-in portion of the membrane indicating the absence of neutrophils, which have egressed beyond the collagen gel. The entire membrane-area was thoroughly scanned to identify any neutrophils present on the porous membrane.
Figure 6. Endothelial cell monolayer disruption due to neutrophil transmigration: (A) Differential decay in the net impedance during neutrophil migration with (red curve) and without (blue curve) the blocking of β1 integrins on neutrophil surface. Impedance values for each individual scan is normalized to its initial value before averaging, to yield a common starting point for comparison. n=3 for each test condition. Error bars represent standard error of the means. (B) The changes in the diffusion of FITC-dextran is monitored during the extravasation of untreated neutrophils (blue curve) or β1 integrin blocked neutrophils (red curve). The increase in the fluorescent signal is more for the untreated neutrophils than the antibody treated highlighting the permeability-regulating effects of antibody treatment. n=5 for blue curve, and n=2 for red curve. Error bars represent standard error of mean.
Figure 7. Quantitative analysis of in situ small molecule permeability: (A) Open top μSiM devices were seeded with HPAECs and grown to confluency. (B) The diffusive properties of 40 kDa FITC-dextran through the monolayer were recorded in the basal compartment using an epifluorescence scope and 40X objective. (C) changes in normalized fluorescence intensity 50 μm from the membrane edge were compared to expected results based on conventional transwell permeability values and a COMSOL simulation of the experimental configuration. Root mean square error (RMSE) represents the deviation of the experimental results from the expected. Cell free represents media only devices, while Native represents HPAEC seeded devices. (D) Additionally, conventional permeability values were determined and compared to values recorded in transwell systems. A paired t-test showed a lack of statistical significance between the two experimental configurations (n=3). Error bars represent standard error of mean.
Figure S1. Assembled μSiM device with tetrapolar electrode arrangement and tubing for fluidic interface.
Figure S2. Setup demonstrating different components of the flow circuit namely the nutrient reservoir to feed fresh media to cells, peristaltic pump to recirculate the media and generate the necessary shear stress, fluidic capacitor to dampen the pulsatile nature of flow, and the µSiM-MVM device with wirings and tubings. The reservoir is exposed to incubator environment via 0.22 µm sterile filter, and TEER wires are connected to breadboard circuit (not shown) for resistance measurements.
Figure S3. Representative impedance scans before seeding HUVECs (blue curve), and 2 days after seeding (24 hours static + 24 hours flow, red curve). Insets show the elevated values of net impedance over the period of 2 days, with different amounts of increments over different ranges of frequency. Inset: Equivalent circuit representing the different biological and electrochemical components contributing to the total impedance as measured using impedance analyzer. ZCPE is the impedance of the ITO-media interface that acts as a constant phase element (CPE). Zcell is the capacitive impedance offered by the cell lipid bilayer while Rmed is the resistance offered by the cell media.
Figure S4. COMSOL modeling of the in situ permeability assay: (A) The center cross-sectional geometry of the open top μSiM was built in COMSOL to exact specifications, with the exception of an addition 10 μm tall block to represent the endothelial monolayer. The diffusion of 40 kDa FITC-dextan through the ‘endothelial monolayer’ into the periphery of the basal channel was modeled, and concentration data was collected 400 μm from the membrane center (50 μm from the membrane edge). (B) Experimentally, a 40X objective focused just off the membrane in the basal channel recorded fluorescence images over time (1 fpm for 10 min). The resulting images were imported into a custom designed MATLAB script in which pixel intensity is averaged along the y-axis, and the resulting values are meshed (C). The progression of fluorescence intensity 50 μm from the membrane edge was then normalized to the intensity of the solution added in the top well and plotted over time (D). These values were matched to COMSOL experiments that represented the minimum deviation from the experimental result (as determined by root mean square error analysis). The permeability values of the ‘endothelial monolayer’ input into the respective COMSOL simulation were recorded as experimental permeability values.

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