The microfluidic PT model altered sodium-dependent reabsorption of glucose analog in response to ouabain.

<p>(A) hRPTEC expressed polarized transport proteins Na⁺/K⁺ ATPase and SGLT2 under co-culture conditions. (B) A schematic representation of experimental conditions. Under all conditions, the transport of a fluorescent glucose analog, 2-NBDG, from the filtrate channel (top) into the vascular channel (bottom) was observed using confocal z-stack and time-lapse microscopy to quantify intensity in the vascular channel. (C) 2-NBDG intensity versus time for one device subjected to all conditions sequentially shows the reduction in active transport due to ouabain administration. (D) The bar graph indicates the mean 2-NBDG intensity in the vascular channel at each condition, measured over ~15 minutes for 3 devices. Data are from 3 replicates of the experiment. Error bars represent standard error of the main effects as computed from the error term in the analysis of covariance model. Introducing ouabain to the vascular channel blocked 2-NBDG transport. The 2-NBDG transport recovered when ouabain was rinsed from the system. A second administration of ouabain again blocked 2-NBDG transport. The tissue recovery and repeat effect of ouabain demonstrates dynamic reabsorptive cell-mediated reabsorption function. * P < 0.0005. Experimental results were verified for 3 independent experiments.</p

An in vitro 3D microfluidic model mimics the reabsorptive barrier of the proximal tubule.

<p>A) In vivo, water and solutes cross an epithelial-endothelial barrier in the reabsorption process from filtrate tubule to the peritubular capillaries. (B) The microfluidic channels overlap to create a filtrate channel (green) in communication with a vascular channel (purple). The cross-sectional architecture (inset) mimics in vivo epithelial-endothelial barrier and generates cell-mediated transport through the membrane. (C) A cross-sectional SEM of the device shows a semi-porous membrane, which serves as a scaffold for the epithelial and endothelial cells and separates the filtrate and vascular channels. (D) The membrane sub-micron ridge/groove topography influences tissue organization and function.</p

hMVEC presence enhances the hRPTEC layer.

<p>(A, B) TJ formation in 7-day cultures of hRPTEC without and with hMVEC in the microfluidic device, respectively. hRPTEC formed a more compact tissue layer with clear TJ formation under hRPTEC/hMVEC co-culture conditions. hRPTEC were labeled with anti-ZO-1 (green) and Hoechst (blue). At least 5 images for each tissue layer and at least three replicate samples were analyzed per group. (C) Average number of hRPTEC/mm² in co-culture conditions is more than double that of hRPTEC-only conditions after 7 days of culture. * P < 0.001. Results were verified for 3 independent experiments. (D) hRPTEC in co-culture conditions have increased mitochondrial activity compared to hRPTEC-only conditions, normalized to cell count. The mitochondrial activity of hMVEC cells in co-culture is negligible. * P = 0.002. Error bars represent standard error of the mean from 3 independent tissue samples.</p

hRPTEC and hMVEC in close-contact co-culture form a physiological PT tissue barrier.

<p>(A) The microfluidic channels overlap to create a renal epithelial filtrate channel in communication with an endothelial vascular channel. (B) A close-contact co-culture of hRPTEC and hMVEC were grown in channels on opposite sides of the membrane. hRPTEC were labeled with anti-ZO-1 (green) and hMVEC with anti-vWF (red). (C, D) Confocal slices of the co-cultured cells show a confluent hRPTEC tissue layer and hMVEC tissue layer with clear ZO-1 (green) and vWF (red) expression, respectively. Each tissue layer exists in the xy plane, but is separated in the z-axis by the membrane. Scale bars: 30 μm. (E) A collapsed xz view of the co-culture stack shows clear separation between the tissue layers and a thicker epithelial tissue layer vs. endothelial layer. (F) A z-profile plot illustrates the change in average intensity expression, normalized to respective blank channel intensity values, of ZO-1 and vWF signals through the 14 μm co-culture 3D tissue stack. The width of the peaks correspond to each cell layer thickness, indicating a cuboidal morphology of the hRPTEC tissue in and a squamous morphology of hMVEC tissue. At least 3 replicate samples were repeated over at least 3 batches of experiments.</p

Confirmation of endothelial monolayer integrity.

<p>The integrity of the endothelial monolayer was confirmed by both fluorescence imaging of the dextran distribution and confocal microscopy of fixed and labeled cells. An intact endothelial monolayer gives rise to an abrupt intensity drop between the channel and the gel region once the fluorescently-labeled dextran is introduced. Three hours after dextran injection, a sharp drop in fluorescence intensity is seen across the endothelial layer demonstrating its function as a barrier to macromolecules (a). Fluorescence intensity is quantified using Matlab (b). The dashed arrow in (a) the location and direction for the quantification.The intensity value drops to 15% of is peak value due to the barrier effect. The endothelial monolayer is located near the 400 µm point on the plot (shown with dashed line). Samples fixed on the third day after cell seeding and stained for VE-cadherin and nuclei (DAPI-blue) exhibit well-defined junctions with no apparent gaps in the confluent monolayer (c). The confocal image shows the front view of the microfluidic device.</p

General schematic of the device.

<p>Microfluidic system consisting of three independently addressable media channels, separated by chambers into which an ECM-mimicking gel can be injected (a). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056910#pone-0056910-g001" target="_blank">Figure 1b</a> shows the inside view of the device with endothelial monolayer (blue) covering the center channel. This channel acts as cell channel where both endothelial cells and cancer cells are introduced to form monolayer and transmigrate respectively (b). The green region indicates the 3D space filled with collagen gel and the pink regions indicate the channel filled with medium. Cancer cells which adhere to endothelial monolayer can extravasate into the collagen gel region as shown in (c).</p

Optimization of tumor cell seeding density.

<p>The tumor cell seeding density was optimized to have only a limited number of tumor cells in ROI while maintaining as many experimental ROIs as possible that contain at least one tumor cell so tumor cell events can be observed. Histograms of number of total tumor cells present in each ROI (250 µm×250 µm×120 µm) show different trends in distribution of tumor cells for three different tumor seeding densities: 20,000 cells/ml, 50,000 cells/ml, and 200,000 cells/ml (a). The average value and the histogram can be used for choosing the optimal tumor seeding condition (b). Seeding density of 50,000 cells/ml was chosen as a compromise between mimicking the low number of tumor cells of the <i>in vivo</i> of extravasation condition and increasing the chance to have at least one tumor cell to analyze in any given ROI. The statistical significance was tested with one way ANOVA (p<0.05).</p