Generation and Initial Characterization of 3D Cortical Collecting Ducts (CCDs)‐on‐a‐Chip

Hydrodynamic forces, including fluid shear stress (FSS), circumferential stretch and drag/torque on apical cilia, regulate ion transport in the CCD of the kidney. Studies aimed at examining pathways and regulation of transepithelial transport in this nephron segment are largely limited to experiments in 2D cell culture monolayers, while renal tubular epithelia grown in their native 3D geometry provide a more physiologically relevant environment. Cell doming is a characteristic unique to epithelial cell monolayers and has previously been described by others to reflect cellular differentiation and vectorial transport of salt and water. We describe the doming characteristics of cells grown in a novel in vitro 3D microphysiological model that recapitulates the in vivo geometry of the CCD. 3D CCD‐like tubules were generated using a pin pullout technique to generate a 500 μm hollow lumen through a fibrin/gelatin extracellular matrix (ECM) enclosed within a polydimethylsiloxane scaffold. The lumen was seeded with mpkCCD cells x 24 hr then continuously perfused with cell culture media via a peristaltic pump in a closed loop circuit at a unidirectional FSS of 0.1 dyn/cm2. By 7 d of culture/perfusion, a confluent monolayer of cells lined the channel. Epithelial integrity was demonstrated using widefield microscopy by retention of a continuously perfused 70 kDa FITC‐dextran (25 μM) in the lumen for 5 hr. Confocal microscopy of 50 μm sections of tubules labeled with target‐specific antibodies revealed ENaC, Na‐K‐ATPase and laminin expression. Cell doming was observed after about 30 minutes of stopping flow (no flow) in all tubules. We quantitatively measured doming characteristics by live‐cell differential interference contrast video microscopy in a 1 mm tubule segment at the inlet of each chip after a 1 hr period of no flow, in the absence (control) or presence of 10 μM benzamil (BZ), an inhibitor of ENaC. Interestingly, domes were observed in only 1 of the benzamil treated tubules. Dome dimensions were measured and volume was estimated using the formula for a segment of a sphere. Data are presented as X ± SEM from individual experiments. Significance of differences was tested by unpaired Student t‐test and statistical significance was defined as P values <0.05. The mean dome volume was ~3.1 ± 0.5 nl in 3 tubules in the absence vs. ~0.04 ± 0.01 nl in the 1 tubule that demonstrated doming in the presence of BZ ( P =0.01). In summary, we generated and initially characterized a 3D CCD‐on‐achip that promises to provide a relevant model for studying transport physiology and addressing a variety of clinically important questions. The mechanism(s) underlying dome formation remains to be determined. Support or Funding Information NIH grants T32DK007757 (JLR), R01DK038470 (LMS) and the NIDDK (Re)Building A Kidney Consortium grant U01DK107350 (LMS). Microscopy was performed at the Microscopy CoRE at the Icahn School of Medicine at Mount Sinai. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .