Supplementary Material

Human Proximal Tubule Cells Form Functional Microtissues  

Jenny A. Prange1, Manuela Bieri1, Stephan Segerer2, Charlotte Burger3, Andres Kaech4, Wolfgang Moritz5 and Olivier Devuyst1

 

1Institute of Physiology, Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Zurich; 2Division of Nephrology, Universitätsspital Zurich, Zurich;3Institute of Anatomy, University of Zurich, Zurich;4Center for Microscopy and Image Analysis, University of Zurich, Zurich;5InSphero 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

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)

NaN3: Sodium azide; DOG: 2-Deoxy-D-glucose

  HK-2 p vs controlHRPTEpiC p vs control p HK-2 vs HRPTEpiC

Baseline 26.4 ± 0.02 87.9 ± 4.4   0.03  

Albumin Competition 8.3 ± 1.4 <0.01 29.3 ± 0.9 < 0.001 <0.01  

Metabolic Inhibition (NaN3, DOG) 7 ± 1.5 < 0.001 26.8 ± 5.2 < 0.001 <0.01  

Cold Inhibition (4°C) 6.2 ± 0.4 0.03 37.8 ± 2.2 < 0.001 <0.01  

  Serum concentration (%)

1000 cpd seeded

Cell density (cpd)

10% FBS supplementation

Co-culture

10% FBS and 1000 cpd

  0.5% 2.5% 10% 500cpd 1000cpd 2000cpd HK-2 + NHDF

Day 1 n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Day 2 n.d. n.d. n.d. n.d. n.d. n.d. 230 ± 6 ***

Day 6 28 ± 5 340 ± 20 *** n.d. n.d. n.d. 300 ± 12 333 ± 7 **

Day 10 55 ± 4 373 ± 13 *** 370 ± 6 *** 333 ± 18 370 ± 6 426 ± 7 ** 424 ± 28

Day 11 95 ± 5 393 ± 37 ** 396 ±12 *** 380 ± 12 396 ± 12 413 ± 18 433 ±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.

HK-2 HK-2 + NHDF p

PCNA/DAPI 62 ± 6 62 ± 7 n.s.

AQP1/DAPI 11 ± 1 7 ± 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

  HRPTEpiC HRPTEpiC + Fibroblasts p

PCNA/DAP 32 ± 8 16 ± 1 n.s.

AQP1/DAPI 3 ± 1 13 ± 1 < 0.001

Megalin/DAPI 25 ± 5 87 ± 5 < 0.001

Cubilin/DAPi 12 ± 4 87 ± 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

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).

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 NaN3/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.

Day 1 Day 10Day 2 Day 6 Day 11

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Suppl. Fig. 1

A

B

Day 1 Day 10Day 2 Day 6 Day 11

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Suppl. Fig. 2

Day 1 Day 10Day 2 Day 6 Day 11H

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Suppl. Fig. 3

Pure HK-2H

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Suppl. Fig. 4

Suppl. Fig. 5

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Suppl. Fig. 6

GravityPLUS™

GravityTRAP™