Fig. 2: Coculture of kidney organoid MACS products with RPTECs.

a Timeline (in days) demonstrating the coculture assay. hiPSCs are plated 23 days prior to the coculture as outlined earlier, followed by RPTECs which are subcultured on day –5. Kidney organoids are harvested upon maturation, dissociated, and mixed in equal portions with RPTECs. The cell mixture is then introduced into the chip. b Brightfield images showing the evolution of LTL+ and cocultured tissues with time. The positive fraction-only cells begin to aggregate from ∼D4 and form separable spheroids by D7 (as indicated by red arrowheads), rendering the device impractical for filtration assessments. Whereas no detachment is observed in the coculture with RPTECs. Scale bar, 200 μm. c Z-intensity projected fluorescent images taken from immunostained samples on D7. DAPI, F-actin and EpCAM are represented in blue, yellow, and green, respectively. Once cocultured with RPTECs at a 50/50 ratio, LTL+ cells blend well and make a confluent tissue layer. Coculturing the LTL– fraction with RPTECs however, does not yield a confluent tissue layer. The tissue partially detaches as indicated by white arrowheads. EpCAM is faintly expressed throughout this tissue and mostly in the RPTECs. Gray dashed lines show the whereabouts of the PToC membrane. Scale bar, 200 μm. d Fluorescent, brightfield and merged images of the cocultured tissue on D14. Top rows show that the channel is covered by RPTECs and LTL+ cells equally in its entirety. High magnification pictures at the bottom row elucidate that the dissociated/sorted cells in the mixture not only form small aggregates but are also distributed almost evenly throughout the coculture tissue. Scale bars are in 1 mm and 100 μm on top and bottom rows, respectively. Dashed lines are to guide the eye.