Fabrication of the kidney organoid-on-a-chip
The kidney organoid-on-a-chip was fabricated using a mold produced by a 3D printer (DP120, WOW3D, Korea) as previously described [22, 23]. Briefly, a 3D printed mold was designed by AutoCAD software and the modeling data were transferred to the 3D printing software. Afterwards, an ultraviolet (UV)-curable resin was cured for the mold fabrication. The resulting mold was washed by isopropyl alcohol (IPA, Sigma Aldrich, USA) and was subsequently dried by air blowing to remove the residual uncured resin. Poly(dimethylsiloxane) (PDMS, Sylgard184, Dow Corning, USA) was mixed in a ratio of 10:1 (monomer: curing agent), degassed, poured onto the fabricated mold, and baked at 80 ℃ for 2 h. The cured PDMS chip was carefully detached from the mold and was subsequently bonded onto the slide glass (Marienfeld, Germany) using O2 plasma treatment (Femto Scientific, Korea). The top side of the chip was then covered by a piece of PDMS mold with the inlet and outlet.
Computational fluid dynamics (CFD) simulation
Before the experiments, the performance of the kidney organoid-on-a-chip was simulated using CFD model to investigate the fluid velocity distribution and shear stress acting on the organoid surface. The microchannel modeling was performed with Autodesk Inventor (Autodesk Inc. USA) 3D CAD software and was subsequently imported to COMSOL Multiphysics 5.5 (Comsol Inc., USA) software to conduct the fluidic flow modeling. The fluid was assumed to be water and the microchannel wall was set to no slip condition. The shape of kidney organoid was modeled as an elliptical sphere with a height of 400 μm, a major axis of 500 μm, and a minor axis of 400 μm. In the microchannel, 10 cylindrical microwells were evenly located and the kidney organoids were occupied in an each well. To determine the effect of microwell dimensions on the fluidic flow velocity profile and the wall shear stress on the kidney organoid surface, the diameter and height of the microwells were defined in the range of 600 μm to 2.1 mm and 100 μm to 2 mm, respectively. We calculated the fluidic flow velocity profile by assuming stationary (steady-state), single-phased laminar flow (SPF), with the inflow rates set to 1, 5, 10, 20, and 30 µL/min, respectively. Within the microchannel, the flow velocity \(u\) is dominated by the homogeneous, incompressible Naiver–Stokes equation and continuity equation:
$$\rho \left(u\cdot \nabla \right)u=\nabla \cdot \left[-p{\rm I}+\tau \right]+F$$
$$\rho \nabla \cdot u=0$$
where \(\rho\) is the fluid density, \(u\) is the velocity field, \(\nabla\) is the divergence, \(p\) is the pressure, \(\tau\) is the Stoke’s stress (\(\tau =\mu (\nabla u+\nabla {u}^{T})\)), \(\mu\) is the dynamic viscosity, and \(F\) is the volume force. Afterward, the shear stress acting on the kidney organoid surface is calculated with the equation \({{\uptau }}_{\text{w}}=\mu \cdot \dot{\gamma }\), where \(\mu\) is the dynamic viscosity and \(\dot{\gamma }\) is the shear rate at the boundary of kidney organoid surfaces.
HPSC culture and kidney organoid differentiation
The kidney organoid differentiation was performed as previously described [24]. In brief, hPSCs were plated at a density of 5000 cells/well on a 24-well plate in mTeSR1 medium (Stem Cell Technologies, Vancouver, Canada) plus 10 µM Y27632 (LC Laboratories, Canada) on glass plates (LabTek) coated with 3% Matrigel (Thermo Fisher Scientific, MA, USA). The medium was exchanged for 1.5% Matrigel in mTeSR1, mTeSR1, RPMI (Thermo Fisher Scientific, MA, USA) plus 12 µM CHIR99021 (Tocris, UK), or RPMI plus B27 supplement (Thermo Fisher Scientific, MA, USA). The cells were fed every 2–3 day to promote kidney organoid differentiation. Kidney organoids were seeded into kidney organoid-on-a-chip on day 16.
Preparation of kidney organoid-on-a-chip
Kidney organoid-on-a-chip was sterilized by autoclaving at 120 °C for 30 min and were subsequently dried in 80 °C oven. The kidney organoid-on-a-chip was coated with 1.5% Matrigel containing 100 ng/mL VEGF for overnight to improve the organoid adhesion. After coating, the kidney organoid-on-a-chip was rinsed three times with Dulbecco’s phosphate-buffered saline (DPBS) before seeding kidney organoids.
Immunostaining
hPSCs-derived kidney organoids cultured within kidney organoid-on-a-chip were analyzed immunocytochemically to confirm the spatial distribution of kidney-related cells. After culturing for 16 days within petri dishes, the cells were retrieved and were subsequently cultured with kidney differentiation medium under a fluidic flow condition in a kidney organoid-on-a-chip for an additional 6 days. The cells cultured in a kidney organoid-on-a-chip were fixed for 30 min with 8% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) at 4 °C. The fixed organoids were blocked in 5% donkey serum (Millipore, USA) plus 0.3% Triton-X‐100/PBS, and incubated overnight in 3% bovine serum albumin (BSA, Sigma Aldrich, MO St. Louis. USA) plus PBS with primary antibodies. Primary antibodies against the following proteins were used to characterize various kidney-related cell types: podocytes (anti-NPHS1, R&D AF4269, 1:500), proximal tubular cells (anti-LTL, Vector Labs FL‐1321, 1:500), and ECs (anti-PECAM1, Abcam ab9498, 1:200). After incubating overnight, kidney organoids were washed with PBS twice. Secondary antibodies (1:500 dilutions, Invitrogen, CA, USA) were applied at room temperature for overnight. Each kidney organoid was washed with PBS and fluorescent images were acquired by using a confocal microscope (Olympus, Japan) after counterstaining with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, Invitrogen, CA).
Nephrotoxicity analysis
Tacrolimus (Sigma Aldrich, MO St. Louis. USA) was made up as a 1.8 mM stock solution in dimethyl sulfoxide (DMSO). The reagent was diluted in a culture medium to make an appropriate final concentration. Briefly, the kidney organoids cultured for 6 days in a kidney organoid-on-a-chip were treated 0, 30, and 60 µM tacrolimus concentration for 24 h. After incubation, the kidney organoids transferred into a 96-well plate. The nephrotoxicity was assessed by using a cell counting kit-8 (CCK-8. Dojindo, JAPAN) according to the manufacturer’s procedure. Finally, the OD at 450 nm with a reference wavelength of 690 nm for each sample was measured using a microplate reader (EL800, Bio-Tek Instruments, Winooski, VT, USA).
Live/dead assay
The viability of tacrolimus-treated kidney organoids within a kidney organoid-on-a-chip was analyzed by using a live/dead assay after 6 days. (Thermo Fisher Scientific, MA, USA). Briefly, 5 mL of DPBS containing 2 µL of calcein AM solution and 10 µL of ethidium homodimer-1 solution was added to kidney organoid-on-a-chip and was then incubated at 37 °C in a 5% CO2 incubator for 40 min. The stained organoids were analyzed by using an inverted fluorescence microscope (Olympus, Japan).
Statistical analysis
The data are expressed as mean ± standard deviation for three times independent experiments. Statistical analysis of the data was performed using the one-way ANOVA or Student’s t-test. Prism 8 (GraphPad, San Diego, CA) was used for all statistical analyses.