Fig. 6: Characterization of the RPTEC layer in the bilayer system using z-intensity profiling of proteins and TEM to show the effect of flow.

a Select cross sectional fluorescent confocal image of the RPTEC/HUVEC bilayer system, immunostained for megalin, illustrating the definition of the relative distance of the protein of interest (e.g. megalin) measured from the center of nuclei. Scale bars are 10 μm. b Representative z-intensity profiles of fluorescent signals obtained by averaging emission intensities of various markers throughout a laser scan area of 0.1 cm² with a Δz (pitch) of 200 nm. Average proximal tubule-specific marker-to-nuclei distance in RPTECs (bilayer) obtained under static and perfused culture conditions for (c) megalin, (d) LTL, and (e) SGLT2; N = 2 independent chips were used to make n = 3 random measurements from each; Error bars indicate standard deviation. Statistically significant differences between data pairs are indicated by asterisks, *, **, ***, ****, for p ≤ 0.05, 0.01, 0.001, and 0.0001, respectively. Select cross-sectional TEM images highlight the appearance of the apical membrane of RPTECs in various culture conditions on D14/d4: (f) single layer under static, (g) bilayer under static, and (h) single layer under perfusion culture conditions. The close-up TEM of (i) reveals a tight junction (yellow arrowhead) formed between two adjacent RPTECs under perfusion culture. j A HUVEC at the opposite side of the membrane. Scale bars are 2 μm. All the micrographs are from the cells fixated on D14/d4. M, mitochondria; N, nucleus; V, vacuole; mem, PET membrane, arrows point to tight junctions, and the dashed lines show cell-cell boundaries. k Quantification of the microvilli length/density and the RPTEC height. We define the villi density as the count of protrusions divided by the length of the cell membrane cross-section periphery, measured from individual snapshots. RPTECs/HUVECs are on D14/d4. Clearly RPTECs in the proximity of HUVECs developed denser apical microvilli, resulting a higher surface area. In addition, the microvilli density distribution is narrower in the bilayer configuration. No significant difference in villi lengths was observed between the single layer and bilayer cases in static culture condition. Flow induced shear stress manifested not only longer but also a larger density of microvilli. For quantification purposes, from a total of N = 3 independent TEM observations per condition, n = 9, 5, and 5 RPTEC micrographs were randomly selected from single layer (static), bilayer (static), and single layer (flow) culture conditions, respectively, to measure villi lengths and densities. To measure cell heights, n = 8 and n = 9 micrographs were examined for static and flow conditions, respectively. Error bars represent the standard deviation of data. l Average distances of basement and apical proteins from the nucleus illustrated for RPTEC-only and coculture tissues developed under flow culture condition. Data obtained by z-intensity profiling of fluorescent images. Statistics are derived in a similar fashion to (c–e).