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Overview of FDSS application: With a focus on EFS assay
1
9th June 2016
Hamamatsu 12th European Functional Drug Screening Symposium
Natsumi KATO
Application Engineer
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Temperature control
Analysis of waveform
High speed acquisition
Electric Field Stimulation (EFS)
expansion Various options
Imaging Plate Reader FDSS series FDSS7000EX FDSS/μCELL
Add 1536,384,96wells
at a time
Read Fluorescence
&
luminescence
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Temperature control
Analysis of waveform
High speed acquisition
Electric Field Stimulation (EFS)
0
10
20
30
40
50
60
0min 5min 10min 20min 30min
With heater (37℃)
0
10
20
30
40
50
60
0min 5min 10min 20min 30min
Without heater (RT)
N=9 N=9
hiP
SC-d
eriv
ed
car
dio
myo
cyte
s B
eati
ng
rate
(/m
in)
FDSS options
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Temperature control
Analysis of waveform
High speed acquisition
Electric Field Stimulation (EFS)
120 ms sampling Interval 9 ms sampling Interval
FDSS options
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Temperature control
Analysis of waveform
High speed acquisition
Electric Field Stimulation (EFS)
FDSS options
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Temperature control
Analysis of waveform
High speed acquisition
Electric Field Stimulation (EFS)
stimulate all 96 wells simultaneously cylindrical electrodes Stimulation voltage is changeable column by column
96-channel electrode array
FDSS options
* Only FDSS/μCELL
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Ion channel assays
P - P Interaction
assays
GPCR assays
Disease model
research
Toxicity assays
FDSS applications
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Ion channel assays
P - P Interaction
assays
GPCR assays
Disease model
research
Toxicity assays
Today’s topics
1. iPSC-derived cardiomyocytes
2. iPSC-derived skeletal muscles 3. H9c2 cell line
4. nanoBRET
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Ion channel assays
P - P Interaction
assays
GPCR assays
Disease model
research
Toxicity assays
Today’s topics
1. iPSC-derived cardiomyocytes
• iPSC-derived cardiomyocytes toxicity assay & EFS availability
• From 2D to 3D culture cells & potential of high resolution camera (well analysis)
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Recent information
…… Our data suggest that the membrane potential and intracellular Ca2 + signal are tightly coupled, supporting the idea that the EAD-like signals reported are the accurate representation of an EAD signal of the cardiac action potential. Finally, the EAD-like Ca2 + signal was well correlated to clinically-relevant concentrations where Torsade de Pointes (TdPs) arrhythmias were noted in healthy volunteers treated orally with some of the compounds we tested, as reported in PharmaPendium®
Ca
MP
+Cisapride
+Cisapride
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Human iPSC-derived cardiomyocyte (Cor.4U) Verapamil (Ca2+ channel blocker)
[Verapamil]
Before adding Verapamil
Control 0.01 μ 0.1 μ 1 μM
10 min After adding Verapamil
1.0 Hz EFS
1.5 Hz EFS
2.0 Hz EFS
10 s
0
500
1000
1500
2000
2500
3000
3500
Control 0.01 uM 0.1 uM 1 uM
[Verapamil]
Amplitude
spontaneous1.0Hz1.5Hz2Hz
Cell: Cor. 4U (Axiogensis) Dye: Cal-520 AM(AATbioquest)
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[E-4031]
Before adding E-4031
Control 0.01 μ 0.1 μ
10 min after adding E-4031
1.0 Hz EFS
1.5 Hz EFS
2.0 Hz EFS
10 s
0
100
200
300
400
500
600
700
800
1.0 Hz 1.5 Hz 2.0 Hz
PW
D8
0
EFS frequency
PWD80 Control0.01 uM0.1 uM
Human iPSC-derived cardiomyocyte (Cor.4U) E-4031 (hERG channel blocker)
Cell: Cor. 4U (Axiogensis) Dye: Cal-520 AM(AATbioquest)
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Information
Application note & protocol
http://axiogenesis.com/images/phocadownload/application_notes/AppNote_Cor4U_FDSSuCELL.pdf
https://cellulardynamics.com/assets/CDI_iCellCardiomyocytes2-FDSS_AP.pdf
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Ion channel assays
P - P Interaction
assays
GPCR assays
Disease model
research
Toxicity assays
Today’s topics
1. iPSC-derived cardiomyocytes
• iPSC-derived cardiomyocytes toxicity assay & EFS availability
• From 2D to 3D culture cells & potential of high resolution camera (well analysis)
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Various 3D cell culture microplate
Corning SUMITOMO BAKELITE
Greiner Kuraray InSphero
Coster 3D PrimeSurface
CELLSTAR® Elplasia™ GravityPLUS™ GravityTRAP™
black wall, clear U bottom
Clear wall, clear U/V bottom
clear wall, clear U/V bottom
black wall, clear bottom
clear wall, clear bottom
384/96 384/96 394/96/6 384/96/24 96
Hydrogel Ultra Hydrophilic polymer
Plasma treatment Hunging drop SureDrop™
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Corning 96 & 384 well spheroid microplates
U shape bottom plate Black plate → low background
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FDSS7000EX Low background
Corning spheroid microplate x FDSS
Cells: iCell cardiomyocyte (CDI) Dyes: Cal-520 AM(AATbioquest)
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inSphero spheroid microplate x FDSS
GravityPlus™ & GravityTRAP™ (inSphero )
Hanging drop Small number of cells
Spheroid !
1mm
Neonatal rat primary cardiomyocytes
Average diameter 217.4um±7.1um Spheroids transferred from GravityTRAP plate to normal flat-bottom black plate
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ROI 24 (Normal) ROI 5
Small ROI is necessary to measure spheroids placed in the edge of the well.
inSphero spheroid microplate x FDSS
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Kuraray spheroid microplate x FDSS
Elplasia™ SQ 200 100 (Kuraray)
Suitable for high-throughput screening SBS 96-, 384-well plate format A number of micro-spaces that are divided by wall
There are thousands of spheroids in a well, each of which beats at each different timing.
This situation results in that the whole-well measurements show no apparent waveform of Ca2+ transients. (Upper picture)
ratio
Well average Cells: Cor. 4U (Axiogensis) Dyes: Cal-520 AM(AATbioquest)
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1 (Well average)
2 (red 1pixel) 3 (green 1pixel)
camera one pixel ≒ one Speroid One pixel data shows a Ca2+ oscillation
of each spheroid
1 s
Kuraray spheroid microplate x FDSS with high-resolution camera (“well analysis”)
Analyzed using Hamamatsu Software, AQUACOSMOS
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Each spheroid in a well beats at each different timing
The beatings of almost all of spheroids
in a well were synchronized by electric stimulations
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Kuraray spheroid microplate x FDSS EFS system
+ EFS
Without EFS With 1.0 Hz EFS With 2.0 Hz EFS Ratio 1.10
1.05
1.00
0.95
Time 0 10 20 30s
0 10 20 30s
0 10 20 30s
3D microplate (Elplasia)
EFS EFS
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Kuraray spheroid microplate x FDSS EFS system
Without EFS With 1.0 Hz EFS With 2.0 Hz EFS Ratio 1.10
1.05
1.00
0.95
Time 0 10 20 30s
EFS EFS
Flat-bottom microplate (2D)
0 10 20 30s
With 2.0 Hz EFS
EFS
3D microplate (Elplasia)
some differences between 2D and 3D 3D cells can be paced at 2 Hz baseline of Ca2+ oscillation
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Ion channel assays
P - P Interaction
assays
GPCR assays
Disease model
research
Toxicity assays
Today’s topics
2. iPSC-derived skeletal muscles 3. H9c2 cell line
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Information
iPS lab (CiRA) Kyoto University, Dr. Sakurai
• They made a Duchenne muscular dystrophy (DMD) cell model induced from patient-derived iPSCs.
• They stimulated their DMD cell model electrically to simulate muscle cell contraction, finding that cells from DMD patients shows the significant increase of Ca2+ influx.
• Such DMD cell models from patient-derived iPSCs have a great potential to develop and evaluate novel drugs for DMD.
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(Lim et al., 2007)
1% FBS
H9c2 cells as a disease model
differentiation
H9c2 cells rattus myoblast Sometimes used as an alternative for cardiomyocytes
L-type Ca2+ channel expression
H9c2 myoblast like myotube
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H9c2 cells as a disease model
10 s
EFS (5V) EFS
EFS EFS
+ 10 μM Nimodipine (Ca2+ channel blocker)
H9c2 myoblast differentiated H9c2 (like myotube) [Ca2+] Ratio
1.15
0.95
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Ion channel assays
P - P Interaction
assays
GPCR assays
Disease model
research
Toxicity assays
Today’s topics
4. nanoBRET
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BRET1 BRET2 nanoBRET™
BRET assay
https://fr.wikipedia.org/wiki/Bioluminescence_Resonance_Energy_Transfer
Promega Technical Manual “NanoBRET™ KRas/BRaf Interaction Assay”
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nanoBRET™
• Very Bright • Small size (19kDa) • High emission intensity • Relatively narrow spectrum
(460 nm peak intensity) • Spectral separation (>175 nm)
nanoBRET assay
(ACS Chem. Biol. 10; 1797, 2015)
Promega Technical Manual “NanoBRET™ KRas/BRaf Interaction Assay”
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NanoBRET™ assay using FDSS luminescence
NanoBRET™ Control Protein Calibration Panel • NanoLuc-HaloTag fusion protein + NanoBRET™ Control protein • 5 types of NanoBRET™ Control protein panel
0%, 0.1%, 1%, 10%, and 100% NL-HT NanoBRET fractional occupancy
• Limit of quantitation (LOQ) represents the minimum percentage of BRET pairs relative to the total donor population
Detection limit of FDSS was validated by using the NanoBRET™ Control Protein
LOQ = slope
10* SD [0% sample]
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NanoBRET™ assay using FDSS luminescence
Detection of LOQ
Detector EM-CCD camera -80℃ (with water cooling OP) 4x4 binning (for 96 well) Camera Exposure time 1 sec (Ave Lumi) Filter 456(Donor), 610 (Acceptor)
R² = 0.9992
1.0E-05
1.0E-04
1.0E-03
1.0E-02
1.0E-01
1.0E+00
0.01% 0.10% 1.00%10.00%100.00%1000.00%
R² = 0.9965
1.0E-04
1.0E-03
1.0E-02
1.0E-01
1.0E+00
0.01% 0.10% 1.00%10.00%100.00%1000.00%
GloMax® FDSS7000EX
LOQ = 0.23378% LOQ = 0.34920%
Data provided by Promega
Detector Photomultiplier tube (PMT)
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Summary
FDSS with the EFS systems would be useful for cardiac toxicity assay and disease model research
FDSS can measure Ca2+ transients in various 3D spheroids of cardiomyocytes
Spatial analysis inside a well in the FDSS data, which is possible using a new software and a high-resolution camera (under development), could provide another useful information in 3D spheroid experiments.
FDSS has the equal LOQ to PMT-based microplate reader in measuring nanoBRET
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Axiogenesis • Ralf Kettenhofen • Felix von Haniel InSphero • Irina Agarkova
Thank you for your attention
Acknowledgements
Kuraray • Masaya Hosoda • Yoko Ejiri Promega • Tsutomu Kudo • Michiko Momoi
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