Fig. 5: Establishment of the non-iPSC bilayer system.

a The timetable of generic cell seeding and maintenance processes. HUVECs are not included for the single-layer devices. b Time course of reported resistances of the RPTEC-only (blue circles), bilayer (red squares), and HUVEC-only (green triangles) tissue layers. For the bilayer devices once HUVECs are introduced on D10, the overall resistance jumps upon formation of a confluent endothelial layer and then starts to decline until reaching steady state on D14/d4. Dashed lines indicate the average resistances obtained over the last 4 days of culture (colored bars) for each case. All the measured values are normalized to that of the RPTEC TEER at steady state, i.e. 60 Ω cm² (blue dashed line). Green and red two-headed arrows indicate, respectively, the reported resistance of the HUVEC-only layer and the resistance increment measured upon addition of HUVECs to the RPTEC-only tissue. A stable bilayer is formed on D14/d4. RPTECs were cultured on N = 6 devices, out of which N = 3 were rendered bilayer upon addition of HUVECs on D10. N = 3 devices were dedicated for the HUVEC-only tissue. c Immunohistochemistry for ZO-1, EpCAM, and megalin in RPTEC tissue of the bilayer system along with fluorescent images of corresponding RFP-tagged HUVECs, showing the evolution of tight junctions, degree of re-epithelialization, and distribution of the albumin transporter, respectively. Tissue activity and integrity is peaked between D14/d4 and D17/d7. Scale bar, 50 μm. d Effect of HUVECs on the intensity of ZO-1 expression; In the presence of HUVECs tight junctions appeared from D14/d4 (green arrowhead) onward and became clearly visible on D17/d7, while in the absence of HUVECs they appeared faintly, even at a considerably higher confocal laser transmissivity. No Triton X was used in this experiment. Scale bars are 100 μm. All fluorescent images are confocal z-intensity projected. Scale bar, 100 μm.