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

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

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