4. - cosmobio.co.jp · 4 respiration, they are predicted to better simulate xenob high 5 0.1 1000...
1
ABSTRACT: Hepatocytes in the liver consume much oxygen for metabolism; thus, in culturing environments with improved respiration, they are predicted to better simulate xenobiotic responses of the living tissue and provide an ideal system for in vitro cytotoxicity analyses. HepG2/C3A cultured in our gas-permeable membrane plates spontaneously formed 3D cell aggregates (3Ds) with 5 or more cells’ height, while those in an ordinary impermeable membrane plates formed essentially flat 2D cell aggregates (2Ds). Cell and nuclear sizes for 3Ds were significantly smaller than those of 2Ds, reflecting near natural dimensions. TMRM probe signal intensities measured by a high-content imaging platform were significantly greater allowing wider analytical ranges for 3Ds than 2Ds. The cells’ responses to FCCP and rotenone were evaluated, and individual plane image analyses were successfully performed in 3Ds. Z’ values generated from the dose response data were typically between 0.4 and 0.7, indicating an analytical robustness. In sum, 3D cell aggregates cultured in the gas-permeable membrane plates can present an ideal cell culture model showing certain physiologically-relevant cell properties for cytotoxicity HCAs. Island-shaped 3D Cell Aggregates on Gas-permeable Membrane Like Islands in the Ocean 3 3 D D R R E E S S P P I I R R A A T T I I O O N N C C U U L L T T U U R R E E S S F F O O R R T T O O X X I I C C I I T T Y Y H H C C A A Jun Kanamune 1 , Hideki Matsunaga 2 , Chongmun Kim, 2 Kazuyuki Yokokawa 2 1 Department of Organ Reconstruction, Field of Clinical Application, Institute for Frontier Medical Sciences, Kyoto University, Kyoto; 2 Bioscience Department, IKKO-ZU Corporation, Tokyo, Japan 60 48 36 24 12 0 m Z stack X-Y image PDMS membrane Air diffusing from underneath Island- shaped 3D structure 1 1 . . Culture Island 3D Cell Aggregates. Quick & Easy 2 2 . . Can Get 3Ds in 2 Days. Tall (Z height) & Compact 37ºC, 5% CO2 Stop dissociation with cold media Dissociate cells by a trypsin -free agent Cell suspension sieved through 40 m mesh Centrifuge once 50 x g Seeded into membrane plates Left undisturbed to have cells setted at bottom Incubated 4-10ºC 4ºC, 2 min RT, 5 min RT, 20 min RT RT Porcine Atelo-Collagen 0 5 10 15 20 25 30 35 40 0 20 40 60 80 100 120 140 0 5 10 15 20 25 30 35 40 Width (m) 0 20 40 60 80 100 120 140 Rat-tail Collagen I 110 0 2 4 6 8 10 12 14 16 10 30 50 70 90 Porcine Atelo-Collagen Number of 3D structures Rat-tail Collagen I 0 2 4 6 8 10 12 14 16 10 30 50 70 90 110 Peak Height (m) 3 3 . . 20 60 90 80 70 Nuclei size (m 2 ) 50 40 30 10 0 Monolayer Island 3Ds 20 m Island 3Ds Give Robust Dose Response Analyses. Wide Emission Range & High Z’ 4 4 . . 5 5 . . Island 3Ds Give Good Image Acquisition. Less Light Absorption & Good Probe Diffusion Day 0 Day 0 100 m 50 m Day 2 50 m 0.1 5 4 3 2 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Neg-6 Neg-5 Neg-4 Neg-3 Neg-2 Z' Concentration ranges Island 3Ds’ Applications Simulate Liver Zonation & Override Crabtree Effect PDMS membrane Well Sealing film Bare partially Bare entirely ★ For inquiry, contact: Jun Kanamune, Ph. D., E-mail: [email protected] or Chongmun Kim, E-mail: [email protected] For information on VeCell gas-permeable membrane plate⇒ http://www.ikko-zu.com/bioscience/ For Acappella Analysis: Ryo Shioda, Ph. D., Senior Application Specialist, Imaging Team, PerkinElmer Japan, E-mail: [email protected] Gas-permeable Membrane Stratified 3Ds Island 3Ds Spheroid Excitation light Emission Light Probe entering Detection limit Detection limit NUCLEI NUCLEI CELLS CELLS 1 2 3 4 Sealed Bare partially Acetamonophen (mM) TMRM Intensity 0 10 20 30 40 50 1.2 10 5 2.5 20 Edge line Bare eintirely Bare partially Island 3Ds TMRM Emission Intensity 0 5 10 15 20 25 30 35 40 45 0.001 0.01 0.1 1 10 100 1000 5 mM Glucose ID 50 = 0.12 M 30 0 5 10 15 20 25 0.001 0.01 0.1 1 10 100 1000 24 mM Glucose ID 50 = 10 M Rotenone (M), 24 hrs 0 5 10 15 20 25 30 35 0.001 0.01 0.1 1 10 100 1000 0 mM Glucose ID 50 = 0.3 M Monolayer 0 4 8 12 16 20 24 28 32 36 0.001 0.01 0.1 1 10 100 1000 24 mM Glucose ID 50 = ? M 0 5 10 15 20 25 30 35 40 0.001 0.01 0.1 1 10 100 1000 0 mM Glucose ID 50 = 0.3 M? 0.001 0.01 0.1 1 10 100 1000 0 5 10 15 20 25 30 35 40 5 mM Glucose ID 50 = ? M Island 3Ds 50 m Monolayer 6 6 . . Detachment of dead cells 0 1 2 3 4 5 6 7 8 9 10 0.01 0.1 1 10 100 1000 TMRM Intensity FCCP (M) 0 0.5 1 1.5 0.01 0.1 1 10 100 1000 TMRM Intensity FCCP (M) -30 -25 -20 -15 -10 -5 0 1000 100 10 1 0.1 Z' FCCP concentration (M) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1000 100 10 1 0.1 Z' FCCP concentration (M)
Transcript of 4. - cosmobio.co.jp · 4 respiration, they are predicted to better simulate xenob high 5 0.1 1000...
ABSTRACT: Hepatocytes in the liver consume much oxygen for
metabolism; thus, in culturing environments with improved
respiration, they are predicted to better simulate xenobiotic
responses of the living tissue and provide an ideal system for in
vitro cytotoxicity analyses. HepG2/C3A cultured in our
gas-permeable membrane plates spontaneously formed 3D cell
aggregates (3Ds) with 5 or more cells’ height, while those in an
ordinary impermeable membrane plates formed essentially flat
2D cell aggregates (2Ds). Cell and nuclear sizes for 3Ds were
significantly smaller than those of 2Ds, reflecting near natural
dimensions. TMRM probe signal intensities measured by a
high-content imaging platform were significantly greater
allowing wider analytical ranges for 3Ds than 2Ds. The cells’
responses to FCCP and rotenone were evaluated, and individual
plane image analyses were successfully performed in 3Ds. Z’
values generated from the dose response data were typically
between 0.4 and 0.7, indicating an analytical robustness. In sum,
3D cell aggregates cultured in the gas-permeable membrane
plates can present an ideal cell culture model showing certain
physiologically-relevant cell properties for cytotoxicity HCAs.
Island-shaped 3D Cell Aggregates
on Gas-permeable Membrane Like Islands in the Ocean
33DD RREESSPPIIRRAATTIIOONN CCUULLTTUURREESS FFOORR TTOOXXIICCIITTYY HHCCAA
Jun Kanamune1, Hideki Matsunaga2, Chongmun Kim,2 Kazuyuki Yokokawa2
1 Department of Organ Reconstruction, Field of Clinical Application, Institute for Frontier Medical
Sciences, Kyoto University, Kyoto; 2 Bioscience Department, IKKO-ZU Corporation, Tokyo, Japan
60 48 36 24 12 0 m
Z stack
X-Y image
PDMS membrane
Air diffusing from underneath
Island- shaped 3D structure
11..
Culture Island 3D Cell Aggregates.
Quick & Easy
22..
Can Get 3Ds in 2 Days. Tall (Z height) & Compact
37ºC, 5% CO2
Stop dissociation with cold media
Dissociate cells by a trypsin -free agent
Cell suspension
sieved through 40 m mesh
Centrifuge once 50 x g
Seeded into membrane plates
Left undisturbed
to have cells setted at bottom
Incubated
4-10ºC
4ºC, 2 min
RT, 5 min
RT, 20 min
RT
RT
Porcine Atelo-Collagen
0
5
10
15
20
25
30
35
40
0 20 40 60 80 100 120 140
0
5
10
15
20
25
30
35
40
Width (m) 0 20 40 60 80 100 120 140
Rat-tail Collagen I
110 0
2
4
6
8
10
12
14
16
10 30 50 70 90
Porcine Atelo-Collagen
Nu
mb
er
of
3D
str
uc
ture
s
Rat-tail Collagen I
0
2
4
6
8
10
12
14
16
10 30 50 70 90 110 Peak Height (m)
33..
20
60
90
80
70
Nu
cle
i s
ize
(
m2)
50
40
30
10
0 Monolayer Island 3Ds
20 m
Island 3Ds Give Robust Dose Response Analyses. Wide Emission Range & High Z’
44..
55..
Island 3Ds Give Good Image Acquisition. Less Light Absorption & Good Probe Diffusion
Day 0 Day 0
100 m
50 m
Day 2
50 m
1000 100 10 1 0.1
5
4
3
2
1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Neg-6 Neg-5 Neg-4 Neg-3 Neg-2
Z'
Concentration ranges
Island 3Ds’ Applications Simulate Liver Zonation
& Override Crabtree Effect
PDMS
membrane
Well
Sealing film Bare partially
Bare entirely
★ For inquiry, contact: Jun Kanamune, Ph. D., E-mail:
Chongmun Kim, E-mail: [email protected]
For information on VeCell gas-permeable membrane plate⇒
http://www.ikko-zu.com/bioscience/ For Acappella Analysis:
Ryo Shioda, Ph. D., Senior Application Specialist, Imaging Team,
PerkinElmer Japan, E-mail: [email protected]
Gas-permeable Membrane
Stratified 3Ds Island 3Ds Spheroid
Excitation light
Emission Light
Probe entering
Detection limit Detection limit
NUCLEI NUCLEI CELLS CELLS
1 2 3 4
Sealed Bare partially
Acetamonophen (mM)
TM
RM
In
ten
sit
y
0
10
20
30
40
50
1.2
5
10 5 2.5 20
Edge line
Bare eintirely
Bare partially
Island 3Ds
TM
RM
Em
issio
n I
nte
nsit
y
0
5
10
15
20
25
30
35
40
45
0.001 0.01 0.1 1 10 100 1000
5 mM Glucose
ID50 = 0.12 M
30
0
5
10
15
20
25
0.001 0.01 0.1 1 10 100 1000
24 mM Glucose
ID50 = 10 M
Rotenone (M), 24 hrs
0
5
10
15
20
25
30
35
0.001 0.01 0.1 1 10 100 1000
0 mM Glucose
ID50 = 0.3 M
Monolayer
0
4
8
12
16
20
24
28
32
36
0.001 0.01 0.1 1 10 100 1000
24 mM Glucose
ID50 = ? M
0
5
10
15
20
25
30
35
40
0.001 0.01 0.1 1 10 100 1000
0 mM Glucose
ID50 = 0.3 M?
0.001 0.01 0.1 1 10 100 1000 0
5
10
15
20
25
30
35
40
5 mM Glucose
ID50 = ? M
Island 3Ds
50 m
Monolayer
66..
Detachment of dead cells
0
1
2
3
4
5
6
7
8
9
10
0.01 0.1 1 10 100 1000
TM
RM
Inte
nsity
FCCP (M)
0
0.5
1
1.5
0.01 0.1 1 10 100 1000
TM
RM
In
ten
sity
FCCP (M)
-30
-25
-20
-15
-10
-5
01000 100 10 1 0.1
Z'
FCCP concentration (M)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1000 100 10 1 0.1
Z'
FCCP concentration (M)
