Cell Culture
Renal proximal tubule epithelial cells (RPTEC; Kidney PTEC Control Cells, SA7K Clone, Sigma, Germany, MTOX1030) were cultured on PureCol-coated (Advanced BioMetrix, 5005-B, diluted with 1:30 in HBSS (Sigma H6648), 20 min incubation at 37°C) T75 flasks in MEME alpha Modification (Sigma, M4526) supplemented with RPTEC Complete Supplement (Sigma, MTOXRCSUP), l-glutamine (1.87 mM, Sigma, G7513), gentamicin (28 μg/mL, Sigma, G1397), and amphotericin B (14 ng/mL, Sigma, A2942). Cells were incubated in a humidified incubator (37°, 5% CO2), and every 2–3 days, the medium was changed. At 90–100% confluency, cells were washed with HBSS (Sigma, H6648), detached with accutase (Sigma, A6964), pelleted (140 g, 5 min), and used for seeding in the OrganoPlate. Cells for experiments were used up to passage 3.
OrganoPlate Culture
For all experiments, a three-lane OrganoPlate (Mimetas BV, 4003 400B) with a channel width of 400 μm and a height of 220 μm was used. 1.6 μL of extracellular matrix (ECM) gel composed of 4 mg/mL collagen 1 (AMSbio Cultrex 3D Collagen I Rat Tail, Cat. 3447-319 020-01), 100 mM HEPES (Life Technologies, 15630), and 3.7 mg/mL NaHCO3 (Sigma, 320 S5761) was injected into the middle inlet (Fig. 1a) of all 40 chips. After a polymerization time of 20 min, 20 μL HBSS was added on top of the collagen 1 and the plate was incubated in a humified incubator at 37°C over night. After polymerization of the ECM, the plate could be also stored in a humified incubator (37°C) for up to a week. RPTEC were detached and resuspended in medium at a concentration of 10 × 106 cells per mL. Two microliters of the cell suspension (20 × 103 cells) was injected into each top inlet, followed by an addition of 50 μL medium to the same well. For control chips, 2 μL of medium was injected into the top inlet instead of the cell suspension. Subsequently, the OrganoPlate was placed for 5 h at an angle of 70° in the incubator (37°C, 5% CO2, humidified). After attachment of the cells, 50 μL of medium was added to the top outlet, bottom inlet, bottom outlet (Fig. 1a), and HBSS on the gel was removed. The OrganoPlate was placed flat in an incubator on an interval rocker platform (± 7° angle, 8 min interval) enabling a bidirectional flow though the perfusion channels (see Fig. 5S). At day 3, antibiotics (gentamycin and amphotericin B) were removed from the medium. The medium was replaced every 2–3 days. Forty-eight-hour toxicant exposures were started at day 6; all other experiments were performed at days 7, 8, 9, or 10. To show the effect of flow in the system, an OrganoPlate was taken off the rocker platform from day 1 to day 4. At day 4, the medium was refreshed and the plate was rocked again under same conditions as the control experiments.
Immunohistochemistry
RPTEC tubes were fixed by replacing the medium with 3.7% formaldehyde (Sigma, 252549) in HBSS (Sigma, 55037C) for 10 min. Tubules were washed with washing solution (4% fetal bovine serum (Gibco, 16140-071) in HBSS) and permeabilized (0.3% Triton X-100 (Sigma, T8787) in HBSS for 10 min. Next, cells were incubated for 45 min in blocking solution (2% FBS, 2% bovine serum albumin (BSA) (Sigma, A2153), and 0.1% Tween 20 (Sigma, P9416) in HBSS). Hereafter, cells were incubated with the primary antibodies, diluted in blocking solution, for 60 min at room temperature. Primary antibodies against Ms-a-ezrin (BD Biosciences, 610602, 1:200), Ms-a-acetylated tubulin (Sigma, T6793, 1:4000), Rb-a-Zonula occludens-1 (ZO-1) (Thermo Fischer, 61-7300, 1:125, rabbit), Rb-a-Phospho-Histone (H2A.X) (Cell Signaling Technology, 9718S, 1:200, rabbit), mouse isotype (Life technologies, 86599), and rabbit isotype (Life technologies, 86199) were used. Subsequently, cells were washed three times with washing solution and then incubated for 30 min at room temperature with secondary antibodies Gt-a-Ms IgG (H+L) Alexa Fluor 555 (Life Technologies, A21422, 1:250), Gt-a-Rb IgG (H+L) Alexa Fluor 488 (Life Technologies, A32731, 1:250) diluted in blocking solution. After washing the tubules three times, nuclei were stained with DraQ5 (Abcam, ab108410, 1:1000) or Nucblue-fixed cell stain (Life Technologies, R37606, 2 drops/mL) or Actin red (Life Technologies, R37112, 2 drops/mL) in the last washing step. Fluorescent images for the 3D reconstructions were taken with the Leica SP5-Sted Confocal Microscope. A z-stack of 220 μm with 2 μm between each image plane was imaged with Alexa 488, Alexa 555, and Alexa 647. Fluorescent images for the analysis of the protein expression after a toxicant exposure were taken with the ImageXpress® Micro Confocal High-Content Imaging System (Molecular Devices). A z-stack of 5 μm between each image plane was imaged for DAPI, FITC, TRITC, and Cy5 channels. A maximum projection was created for depicting the images, and a summary projection was used for quantifying the fluorescent intensity of the markers.
Barrier Integrity Assay
The barrier integrity assay (BI assay) was performed by replacing the medium of the perfusion channel with medium containing 0.5 mg/mL TRITC-dextran (4.4 kDa, Sigma, FD20S) and 0.5 mg/mL FITC-dextran (155 kDa, Sigma, T1287). Next, the plate was imaged every 2 min for 12 min with the ImageXpress Micro XLS-C High Content Imaging System (Molecular Devices) at 37°C. Leakage of the dyes from the apical side of the tube to the basal side into the ECM was measured, and the ratio between the basal and the apical was analyzed with Fiji (13). The labeled dextrans can be washed out after each measurement. The permeability of the membranes was analyzed by measuring the amount of molecules which leaked though the membrane into the adjacent gel lane over time. From these measurements, the apparent permeability index (Papp: initial flux of a compound through a membrane, normalized by membrane surface area and donor concentration) was calculated by the following formula:
$$ {P}_{\mathrm{app}}=\frac{\Delta {C}_{\mathrm{receiver}}\times {V}_{\mathrm{receiver}}}{\Delta \mathrm{t}\times {A}_{\mathrm{barrier}}\times {C}_{\mathrm{donor}}}\ \left(\frac{cm}{s}\right) $$
∆Creceiver is the measured normalized intensity difference of the ECM to the donor channel (Fig. 2b value of D/value of C) at t0 min and t10 min, Vreceiver is the volume of the measured region in the ECM channel (Fig. 2b, c; channel height × channel width × channel length = 220 μm × 2304 μm × 204.8 μm = 0.0001 cm2), ∆t is the time difference t10 min − t0 min = 10 min, Abarrier (0.0057 cm2) is the surface of the ECM interface with the medium channel, and Cdonor is the donor concertation of the dextran dye (0.5 mg/mL) (Fig. 2c).
Cisplatin Exposure
To determine the toxic effect of cisplatin on RPTEC tubules in the OrganoPlate, medium of both channels (apical and basal) was replaced at day 6 after seeding with TOX medium (MEME alpha Modification (Sigma, M4526) supplemented with RPTEC Tox Supplement (Sigma, MTOXRTSUP), l-glutamine (1.87 mM, Sigma, G7513)) in the presence of 0, 5, 15, 30, 90, 135, or 270 μM cisplatin (Sigma, P4394, stock: 5 mM in 0.9% NaCl (Sigma, S7653) in H20). After 48-h incubation on the rocker platform, phase contrast images were taken and the medium was sampled from the top channel. Samples from in- and outlet were pooled and used for the LDH activity assay. Next, tubes were incubated with WST-8 to determine cell viability. The barrier integrity of the exposed tubules was assessed consecutively of the WST-8 assay. After the exposures and viability measurements, the tubules were fixed with formaldehyde and stained with H2A.X, actin, and ZO-1.
Lactate Dehydrogenase Activity Assay
Lactate dehydrogenase (LDH) activity of the samples was determined using the Lactate Dehydrogenase Activity Assay Kit (Sigma, MAK066) according to the manufacturer protocol. In short, the medium of the top in- and top outlet was pooled and 2 μL was added in duplicate to a 384 well plate. In parallel, a concentration curve of the NADH standard was added. Next, 18 μL LDH Assay Buffer was added to all sample wells to bring to an initial volume of 20 μl. After a short centrifugation of the plate, 20 μl Master Reaction Mix were added to each well and mixed on a horizontal shaker in the plate reader. After 1 min, the absorbance was measured at 450 nm. While the plate was incubated, it was measured every 2 min until the value of the most active sample was higher than that of the highest standard (12.5 nmol/well). For the analysis, the LDH activity was determined using the following formula
$$ \mathrm{LDH}\ \mathrm{activity}=\frac{B\times \mathrm{sample}\ \mathrm{dilution}\ \mathrm{factor}}{\left(\mathrm{reaction}\ \mathrm{time}\right)\times V} $$
where B is the amount (nanomole) of NADH generated between tinitial and tfinal, the reaction time is tfinal − tinitial (in minutes), and V is the sample volume (in milliliters) added to the well.
Cell Viability (WST-8 Assay)
The cell viability of the cells was determined using the Cell Counting Kit—8 (Sigma, 96992). The WST-8 solution was diluted 1:11 with TOX medium and added to the channels of the OrganoPlate (30 μL in- and outlets). After 18 min on the rocker platform and a 2-min flat incubation, the absorbance in the top in- and outlets was measured with the Multiskan™ FC Microplate Photometer (Thermo scientific) at 450 nm.
Calcein-AM Efflux Inhibition
Medium in all perfusion channels was replaced with 1 μM calcein-AM (Life technologies, C3099, stock: 1 mM in DMSO) in KHH buffer (Krebs-Henseleit (Sigma, K3753) + 10 mM HEPES (Gibco, 15630) adjusted to pH 7.4) in the presence of 10 μM cyclosporin A (Sigma, 30024, stock 5 mM in DMSO), 500 μM Digoxin (Fluka, 4599, stock 100 mM in DMSO), or 0.5% DMSO (Sigma, D8418, vehicle control). After a 60-min incubation on the rocker platform, chips were washed one time with ice cold KHH buffer. In the next washing step, Hoechst 33342 (2 drop/mL, Life Technologies, R37605), 10 μM PSC833 (Sigma, SML0572, stock: 5 mM in DMSO), 10 μM Ko143 (Sigma, K2144, stock 10 mM in DMSO), and 10 μM MK571 (Sigma, M7571, stock 10 mM in H2O) were added to the washing solution and the plate was imaged with the ImageXpress® Micro Confocal High-Content Imaging System.
MRP2/4 Efflux Inhibition
Medium in all perfusion channels was replaced with 1.25 μM CMFDA (Molecular Probes, C7025, stock 2.5 mM in DMSO) in the presence of 0, 10, 20, and 30 μM MK571 (Sigma, M7571, stock 10 mM in H2O) in KHH buffer. After a 30-min incubation on the rocker platform, the chips were washed one time with ice cold KHH buffer. In the next washing step, Hoechst 33342 (2 drops/mL, Life Technologies, R37605), 10 μM PSC833 (Sigma, SML0572, stock 5 mM in DMSO), 10 μM Ko143 (Sigma, K2144, stock 10 mM in DMSO), and 10 μM MK571 (Sigma, M7571) were added to the washing solution and the plate was imaged with the ImageXpress® Micro Confocal High-Content Imaging System.
6-NBDG Influx Inhibition
Medium in the apical channel (Fig. 1) was replaced with OptiHBSS (1/3 Opti-MEM (Gibco, 11058-021), 2/3 HBSS (Sigma, H6648)) containing 500 μM 6-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-Deoxyglucose (6-NBDG, Molecular Probes, N23106, lot 1704487, stock 10 mM in H2O) and 0, 20, 100, or 500 μM Phlorizin (Sigma, P3449, stock 200 mM in Ethanol (JT Baker, 8025.2500PE)). All conditions contained 0.25% ethanol as vehicle. Medium in the basal channel was replaced with 6-NBDG-free medium, concentrations of phlorizin matched apical channel concentrations. After a 30-min incubation on the rocker platform, cells were washed two times with ice cold OptiHBSS. In the second washing step, Hoechst 33342 (2 drops/mL, Life Technologies, R37605) was added to the washing solution and the plate was imaged with the ImageXpress® Micro Confocal High-Content Imaging System.
Image Acquisition and Analysis of the Transport Experiments
For the in-cell transport assays, plates were imaged with the ImageXpress® Micro Confocal High-Content Imaging System. A z-stack of 220 μm with 10 μm between each image plane was imaged with the FITC and the DAPI channel (Fig. S1). The intensity of the FITC signal of the cells growing against the ECM was analyzed with Fiji (13) and corrected for the background and cell number. Treated chips were normalized against vehicle control.
Transepithelial Transport Assay
Medium in the apical channel was replaced with medium containing 20 μM cyclosporin A or 0.4% DMSO. Medium in the basal channel was replaced with TOX medium containing 10 μM rhodamine 123 (Sigma, 83702, stock 50 mM in ethanol) together with 20 μM cyclosporin A or 0.4% DMSO. To determine the concentration of rhodamine 123, a concentration curve was added to unused chips. Tubules were incubated for 5 h on the rocker platform. After 3 h and after 5 h, rhodamine 123 concentration was measured by imaging the top inlets with the FITC filter on the ImageXpress Micro XLS-C High Content Imaging Systems. The apparent permeability (Papp) was calculated by using the formula
$$ {P}_{\mathrm{app}}=\frac{\Delta {C}_{\mathrm{receiver}}\times {V}_{\mathrm{receiver}}}{\Delta \mathrm{t}\times \mathrm{A}\times {C}_{\mathrm{donor}}}\ \left(\frac{\mathrm{cm}}{\mathrm{s}}\right). $$
Creceiver is the measured intensity difference in the top wells between t3 h and t5 h, Vreceiver is the receiving volume in the reservoirs of the top inlets, t is the time difference t5h − t3h = 2 h, A is the surface of the ECM interface with the medium channel, and Cdonor is the donor concertation of 10 μM rhodamine 123.
Flow Simulation and Experimental Verification
The platform described in this work uses a gravity-based perfusion system. The fluid flow rate and induced shear stress in the microfluidic channels of the OrganoPlate was estimated using a numerical model simulated in Python (Python Software Foundation, USA). This model calculates the induced pressure difference between two volumes of fluid, which are present in two microtiter plate wells that are connected by a microfluidic channel. The numerical model is described in more detail in the supplementary information. To validate the numerical model of the gravity-driven flow in the OrganoPlate, absorption was sequentially measured at 494 nm using a Fluorescein solution (Sigma, 46960, 10 μg/mL in water). For the verification, a 9603200B OrganoPlate (two-lane plate with 120 × 200 μm, w × h channels) was used. The FITC solution was added to the channel system with 50 μL in each in- and outlet. After tilting the plate at a set angle, the fluorescence of both wells was measured and compared with the associated simulated volumes.
Statistics and Data Analysis
Images were analyzed using Fiji (13). Data analysis was performed using Excel (Microsoft office 2016) and GraphPad Prism (GraphPad Software Inc., version 6.07). Error bars represent the standard deviation. Data were analyzed using one-way ANOVA followed by a Dunnett multiple comparison test which compares all treated chips to the control chips. Comparisons of two groups were done using the t test. A p value of < 0.05 was considered to be significant. At least three technical replicates per data point were obtained.