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. Author manuscript; available in PMC: 2022 Mar 5.
Published in final edited form as: Sci Transl Med. 2021 Feb 24;13(582):eabd6299. doi: 10.1126/scitranslmed.abd6299

Fig. 2. Characterization of nephrotoxicity in sensor-embedded vascularized kidney spheroids.

Fig. 2.

(A) Confocal microscopy of kidney spheroids composed of EGFP-labeled microvascular endothelial cells and aquaporin 1–positive (AQP1+) proximal tubule cells after 4 days in culture. Scanning electron micrograph of vascularized kidney spheroid shown on right. Arrows indicate patent endothelial lumens. Scale bars, 10 μm. (B) Confocal microscopy of proximal tubules expressing apical surface LTL in a spheroid. 3D reconstruction shows parallel longitudinal tubule-like structures 240 ± 40 μm in length. (C) Gene expression of RNA-seq data from human embryonic kidney (HEK) 293 cells, human kidney 2 (HK2) cells, and primary proximal tubule cells (hPTCs) in 2D culture compared to vascularized kidney spheroids and human proximal tubule tissue. (D) Principal component analysis (PCA) of gene expression patterns of vascularized kidney spheroids compared to HEK293, HK2, hPTCs, and proximal tubule tissue in vivo. (E) 3D design of a computer numerical control–fabricated nine-microwell bioreactor. Laser-cut disposable microwell chips containing nine spheroids are seeded in an open configuration and perfused until metabolic stabilization is achieved. (F) Fluorescent microscopy shows a human kidney spheroid with embedded oxygen sensors (orange). Scale bar, 100 μm. (G) Representative oxygen uptake over time response of kidney spheroids exposed to increasing concentrations of cyclosporine or (H) cisplatin. Dotted line notes onset of drug exposure. (I) Time to onset of response of kidney spheroids exposed to cyclosporine or (J) cisplatin. (K) Analytical derivation of nephrotoxic threshold (NT) using the time-to-onset–dependent flux accumulation equation. NT∞ was defined as the horizontal asymptote, concentration for which the onset of damage will only occur at infinite time. Cyclosporine and (L) cisplatin showed NT of 4.9 ± 0.1 nM, a 1000-fold lower than clinically reported Cmax, and 2.8 ± 0.05 μM, respectively. Scale bars, 50 μm. N = 9. N = 3. Error bars indicate ± SE. For RNA-seq analysis, P values from a negative binomial Wald test are reported.