1. Supplementary Material Human Proximal Tubule Cells Form Functional Microtissues Jenny A. Prange 1, Manuela Bieri 1, Stephan Segerer 2, Charlotte Burger 3, Andres Kaech 4, Wolfgang Moritz 5 and Olivier Devuyst 1 1 Institute of Physiology, Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Zurich; 2 Division of Nephrology, Universitätsspital Zurich, Zurich; 3 Institute of Anatomy, University of Zurich, Zurich; 4 Center for Microscopy and Image Analysis, University of Zurich, Zurich; 5 InSphero AG, Zurich, Switzerland Correspondence: olivier.devuyst@uzh.ch, Phone: 044 635 50 82 Supplementary Tables 1 to 4 Legends to Supplementary Figures Supplementary Figures 1 to 6 Supplementary Movie 1

2. Suppl. Table 1. Uptake of Alexa-488 BSA (in ng/µg protein) in HK-2 and HRPTEpiC monolayers. N=3 filters per condition; Alexa-488 BSA, bovine serum albumin (0.5mg/ml, 15min incubation) NaN 3 : Sodium azide; DOG: 2-Deoxy-D-glucose HK-2p vs control HRPTEpiCp vs control p HK-2 vs HRPTEpiC Baseline26.4 ± 0.02 87.9 ± 4.4 0.03 Albumin Competition8.3 ± 1.4<0.0129.3 ± 0.9< 0.001<0.01 Metabolic Inhibition (NaN 3, DOG)7 ± 1.5< 0.00126.8 ± 5.2< 0.001<0.01 Cold Inhibition (4°C)6.2 ± 0.40.0337.8 ± 2.2< 0.001<0.01

3. Serum concentration (%) 1000 cpd seeded Cell density (cpd) 10% FBS supplementation Co-culture 10% FBS and 1000 cpd 0.5%2.5%10%500cpd1000cpd2000cpdHK-2 + NHDF Day 1n.d. Day 2n.d. 230 ± 6 *** Day 628 ± 5340 ± 20 ***n.d. 300 ± 12333 ± 7 ** Day 1055 ± 4373 ± 13 ***370 ± 6 ***333 ± 18370 ± 6426 ± 7 **424 ± 28 Day 1195 ± 5393 ± 37 **396 ±12 ***380 ± 12396 ± 12413 ± 18433 ±18 Average diameter in µm. The microtissues were grown on GravityPlusTM plates and harvested after 12 days. N = 3 samples per culture condition; n.d. sphere not detectable; cpd, N cells per drop ** p<0.01; *** p<0.001, 2.5% and 10% serum concentrations vs. 0.5%; cell concentration vs 1000cpd; Mono- vs Co-culture. Suppl. Table 2.. Diameter of 3D microtissues obtained from HK-2 cells: influence of serum, cell density and co-culture conditions.

4. HK-2HK-2 + NHDFp PCNA/DAPI62 ± 662 ± 7n.s. AQP1/DAPI11 ± 17 ± 1< 0.05 Suppl. Table 3. Analysis of proliferation and differentiation markers (% of positive cells/total cell number) in microtissues from HK-2 in mono-culture and co-culture with fibroblasts. The counts were done on at least 3 sections from 9 microtissues; NHDF, normal human dermal fibroblasts; n.s. not significant HRPTEpiCHRPTEpiC + Fibroblastsp PCNA/DAP32 ± 816 ± 1n.s. AQP1/DAPI3 ± 113 ± 1< 0.001 Megalin/DAPI25 ± 587 ± 5< 0.001 Cubilin/DAPi12 ± 487 ± 20< 0.001 Suppl. Table 4. Analysis of proliferation and differentiation markers (% of positive cells/total cell number) in microtissues from HRPTEpiC in mono-culture and co-culture with fibroblasts. The counts were done on at least 3 sections from 9 microtissues; n.s. not significant

5. Legends to Supplementary Figures: Suppl. Fig. 1. Effect of different serum concentrations on microtissue formation with HK-2 cells. HK-2 cells (1000 cells per drop) were seeded in DMEM-F12 medium supplemented with 0.5%, 2.5% or 10% FBS on GravityPLUS  plates and harvested after 12 days. Representative images are shown (A). The diameter of the microtissues was analyzed using a Bürker chamber (N=3 per condition, B). Higher serum concentration enhances sphere formation, starting from day 6. While 0.5% FBS cultures are still loose and build multiple small spheres, 2.5% FBS cultures start to form irregular aggregates after 6 days. 10% FBS supplemented cultures form regular round spheres after 10 days. Suppl. Fig. 2. Effect of different cell densities on microtissue formation with HK-2 cells. HK-2 cells (500, 1000 and 2000 cells per drop, cpd) were seeded in DMEM-F12 medium supplemented 10% FBS on GravityPLUS  plates and harvested after 12 days. Representative images are shown (A). The diameter of the microtissues was analyzed using a Bürker chamber (N=3 per condition, B). Higher cell density enhances sphere formation. While 500 cpd build microtissue after 11 days, 1000 cpd show round spheres one day earlier and 2000 cpd after 6 days. Suppl. Fig. 3. Co-culture with fibroblasts accelerates HK-2 microtissue formation. HK-2 cells (1000 cpd) were seeded alone or with human fibroblasts (ratio fibroblasts:HK2 1:10 per drop) in DMEM-F12 medium supplemented with 10% FBS on GravityPLUS  plates and harvested after 12 days. Representative images of sphere growth are shown (A). Using a Bürker chamber the diameter of the microtissues was determined (B). Microtissues in co- culture form microtissues already after 2 days and are then growing in diameter until harvest. Suppl. Fig. 4. Characterization of HK-2 microtissues in mono and co-culture. The quality of HK-2 microtissues after 12 days of mono- or co-culture was assessed by hematoxylin and eosin (H&E) as well as immunofluorescence. H&E staining shows dense and compact tissues in both cases, with signs of high proliferation (PCNA). A slight but significant decrease in the differentiation marker AQP1 is observed in co-culture compared to mono- culture conditions. (scale bars 200µm).

6. Suppl. Fig. 5. Morphological characterization of HRPTEpiC microtissues. Representative images of 12 day old pure HRPTEpiC microtissues obtained by transmission electron microscopy. A complete microtissue is shown in panel (A) (scale bar 50µm). Magnified Panels illustrate the microvilli and subapical region (B, scale bar 2µm); the luminar spaces (C, scale bar 10µm); the tight junction complexes (D, scale bar 200nm); and the endolysosomal vesicles (E, scale bar 1µm). Suppl. Fig. 6. Hanging drop cell culture plates. Cell suspensions were seeded in GravityPLUS  plates to form microtissues under gravity-supported conditions. After successful sphere formation, microtissues were transferred into GravityTRAP  plates for long-term cultures and functional assays. A non- adherent surface coating prevents cell attachment and spreading. Suppl. Movie 1. Z-Stack analysis of the endocytic uptake of Alexa 488-Albumin in co-culture HRPTEpiC microtissues under control and metabolic inhibition conditions. Microtissues (control conditions or pre-treatment with NaN 3 /DOG) were incubated with 0.5mg/ml Alexa-Albumin for 1 hour. After washing and PFA-fixation (4%), confocal Z-stacks were taken using a CLSM Leica SP5 Mid UV-VIS.

7. Day 1Day 10Day 2Day 6Day 11 Pure HK-2 0.5% FBS Pure HK-2 2.5% FBS Pure HK-2 10% FBS 200µm n.d. HK-2 2.5% FBS HK-2 10% FBS HK-2 0.5% FBS Diameter of spherical structures (µm) D1D2D6 D10 D11 ** *** n.d. = not detectable Suppl. Fig. 1 A B

8. Day 1Day 10Day 2Day 6Day 11 Pure HK-2 500cpd Pure HK-2 1000cpd Pure HK-2 2000cpd 200µm A ** Diameter of spherical structures (µm) D1D2D6 D10 D11 HK-2 1000cpd HK-2 2000cpd HK-2 500cpd n.d. n.d. = not detectable B Suppl. Fig. 2

9. Day 1Day 10Day 2Day 6Day 11 HK-2 + NHDF Pure HK-2 A Co-Culture Mono-Culture Diameter of spherical structures (µm) D1D2D6 D10 D11 n.d. B n.d. = not detectable *** ** Suppl. Fig. 3

10. Pure HK-2 H&E PCNA/DAPI HK-2 + NHDF AQP1/DAPI positive cells/total nuclei count (%) PCNA AQP1 Mono-Culture Co-Culture * Suppl. Fig. 4

11. Suppl. Fig. 5 AD C B E

12. Suppl. Fig. 6 GravityPLUS™ GravityTRAP™