Post on 19-Jan-2016
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
Pu
re H
K-2
0.5
% F
BS
Pu
re H
K-2
2.5
% F
BS
Pu
re H
K-2
10
% F
BS
200µm
200µm
200µm
n.d
.
n.d
.
n.d
.
050
100150200250300350400450
HK-2 2.5% FBS
HK-2 10% FBS
HK-2 0.5% FBS
Dia
me
ter
of
sph
eric
al
stru
ctu
res
(µm
)
D1 D2 D6D10 D11
*****
******
***
n.d. = not detectable
Suppl. Fig. 1
A
B
Day 1 Day 10Day 2 Day 6 Day 11
Pu
re H
K-2
50
0cp
dP
ure
HK
-21
00
0cp
dP
ure
HK
-22
00
0cp
d
200µm
200µm
200µm
A
050
100150200250300350400450 **
Dia
me
ter
of
sph
eric
al
stru
ctu
res
(µm
)
D1 D2 D6D10 D11
HK-2 1000cpd
HK-2 2000cpd
HK-2 500cpd
n.d
.
n.d
.
n.d
.
n.d. = not detectable
B
Suppl. Fig. 2
Day 1 Day 10Day 2 Day 6 Day 11H
K-2
+ N
HD
FPu
re H
K-2
A
0
50
100
150
200
250
300
350
400
450
500Co-Culture
Mono-Culture
Dia
met
er o
f sph
eric
al s
truc
ture
s (µ
m)
D1 D2 D6D10 D11
n.d
.
n.d
.
n.d
.
B
n.d. = not detectable
***
**
Suppl. Fig. 3
Pure HK-2H
&E
PC
NA
/DA
PI
HK-2 + NHDFA
QP
1/D
AP
I
010203040506070
po
sitiv
e c
ells
/to
tal n
ucl
ei c
ou
nt
(%)
PCNA
AQP1
Mono-Culture
Co-Culture
*
Suppl. Fig. 4
Suppl. Fig. 5
A D
C
B
E
Suppl. Fig. 6
GravityPLUS™
GravityTRAP™