Cardiomyocyte in-vitro Toxicity Assay

Cardiomyocyte
in-vitro Toxicity Assay
iPS
derived cell drug discovery in
FDSS
Recently, studies of iPS cells (induced pluripotent stem cells) have made a huge impact in the drug
discovery field. Currently, human iPS cell (hiPSC) derived various specific types of cells such as
cardiomyocytes and neural cells are now widely available commercially, and the screening of chemical
compounds for drug discovery using these hiPSC-derived is possible. Screening using hiPSC-derived
cells is expected to provide more effective and easy way to evaluate the pharmacological and toxic
effects of test compounds in cell-based assays.
HAMAMATSU has developed new functions for the FDSS/µCELL which allows the measurement and
analysis of calcium transients in hiPSC-derived cardiomyocytes. This is useful for in vitro toxicity
screening using human cardiomyocytes, particularly at the early stage of drug development.
®
FDSS /µCELL
The FDSS/µCELL is a kinetic plate reader with an integrated dispensing head and imaging-based detector. Simultaneous
dispensing into the entire 96/384 well plates and simultaneous detection of the kinetics of the fluorescence or luminescence
intensity allow quick measurements with no time lag for the 96/384 well plate. The technologies employed in the FDSS series are
integrated into a compact body, enabling simple-to-use operation, suitable for assay development or in researching basic cellbased kinetic assay.
Feature
●Small footprint, affordable, easy-to-use
●Simultaneous dispense and imaging whole
96/384 plate
●Dedicated optics to measure all well
uniformly
●Long life and stable LED light source
●2 wavelength measurement options
[FDSS/µCELL components]
Dispenser head
Disposable tips
Assay plate
Excitation
light source
Emission filter
Camera lens
Ca2+-transient measurements in human
iPS-derived cardiomyocytes
Cell: iCell® Cardiomyocytes (Cellular Dynamics International)
Probe: Fluo-8/AM
FDSS/µCELL is capable of measuring Ca2+-transients in iPS/ESderived cardiomyocytes in 96/384-well plate format.
2
Camera (sensor)
FDSS/µCELL
New Option 1
New Functions
●Temperature control with Heater Unit for stable beating
of cardiomyocytes
●High speed data acquisition to accurately measure
calcium oscillation (calcium transients) in
cardiomyocytes.
●Software for analysis of calcium oscillation waveforms
Above three options are developed to have more reliable results from the cardiomyocyte assay. Equipping
with all of these options provide efficiency to compound toxicity study in early drug discovery stage.
New Option 2
New Option 3
Heater unit
High Speed Data
Acquisition option
Analysis Software
for waveform
of calcium oscillation
N=9 well
N=9 well
60
60
50
50
40
40
30
30
20
20
10
10
0
0
5min 10min 20min 30min
The High Speed Data Acquisition option for the
FDSS/µCELL can acquire images with very
short interval times (approx. 10 ms). To
accurately measure the calcium oscillation in
cardiomyocytes requires such high speed.
100 ms
After measuring the calcium oscillation in
cardiomyocytes with the FDSS/µCELL, you
need to analyze the data. The new FDSS
analysis software allows quick and easy
analysis of the waveform of calcium
oscillation.
8 ms
with heater(37 °C)
without heater(RT)
Beating rate(bpm)
New Option 3
New Option 1
The Heater Unit is designed to maintain a
stable temperature of all wells in a
microplate at +35 °C to +37 °C. The beating
of iPSC-derived cardiomyocytes is very
sensitive to temperature and easily looses
stability at room temperature. The heater unit
greatly improves the stability of beating.
0min
New Option 2
Cells: Cor.At® cardiomyocytes (Axiogenesis AG)
0min
5min 10min 20min 30min
Cell: iCell ® Cardiomyocytes (Cellular Dynamics International)
The above graph shows the changes in the
beating rate of human cardiomyocytes in a
microplate on the FDSS/µCELL during 30
minute incubation. Without the heater unit
(left column, at room temperature), the
beating rate gradually decreased with time
and the rate dropped by half after 30
minute incubation. In contrast, when the
well temperature was maintained at +37
°C using the heater unit (right), the beating
rate was unchanged even after 30 minute
incubation.
The above graph shows the fluorescent intensity
change (calcium concentration change) in
cardiomyocytes in a well, which were measured
with 100 ms (left) and 8 ms (right) sampling
intervals respectively. The main difference
between the measurements with the two
sampling rates is the time from the resting
calcium concentration level (bottom) to reaching
to the maximum calcium concentration (peak). It
is shorter when measured with 8 ms intervals,
which shows you may miss the accurate peak
point in measurements with 100 ms sampling
intervals. Shorter sampling intervals enables us
to measure calcium oscillation more accurately.
Above is the capture of the beat
analyzing software. This software is
launched from FDSS software. Open the
data with FDSS software and show the
range to analyze. Then press the button
to launch this software. 16 parameters
can be analyzed by this software.
3
Analysis Software for waveform of calcium
oscillation in cardiomyocytes
Support your analysis with multiple parameters (e.g. peak number) of calcium
oscillation in iPS/ES-derived cardiomyocytes.
Feature
●Visualize and analyze the calcium oscillation in cardiomyocytes.
●Auto-setting and visualized setting configuration.
●Flexible settings for various type of waveform.
●16 analysis parameters available.
Parameters
(1)
Peak number (Total, BPM)
(2)
P-P time [ms] (Ave, Std, Max, Min)
(3)
Ratio (Ave, Std)
(4)
AMP (Ave, Std)
(5)
RMP (Ave, Std)
*Ratio = (AMP + RMP) / RMP
Slope (Ave, Std)
(6)
(7)
Rising Slope: Slope from bottom to peak
Falling Slope: Slope from peak to bottom
peak bottom setting can be selected
0 % - 100 %, 10 % - 90 %, 20 % - 80 %, 30 % - 70 %
Integration (Ave, Std)
(8)
to PWD (PWD10 to 90) [ms] (Ave, Std)
(16)
Fig1
Procedure
data loading
1 FDSS
Press the button in the
FDSS
software to launch the analysis
software and the data shown in
current range is transfered to be
analyzed.
preparation
2 Data
●Interpolation
Duplicate the sampling number to
interpolate the missing sampling for
low sampling rate data.
●Smoothing
Smoothing is to remove the noize in
the waveform data, useful for very
high sampling rate data.
settings
3 Analyze
●Irregular peak detection level
Threshold to judge irregular beating
●Base line
Select median or bottom value to
create base line, and its range.
●Peak parameter
Configure the threshold level from the
baseline to detect peak, and to set the
width range for one peak.
●AUTO
Automatic settings are available.
4 Analyze
Select the necessary
parameter and
analyze for the whole plate or well by
well
Fig2
5 Results
Displays data
in plate format,
waveform for the selected well
●Analyzed data display in plate
format
●Colored plate to visualize the data
value high/low among the wells.
●Analyzed waveform display for the
selected well
out results
6 Text
Exporting data in text file with plate
format, and the data of each well.
4
FDSS/µCELL
Experimental Protocol
Standard protocol for calcium ion assay using iPS-derived
cardiomyocytes are determined by the cell manufacturer.
Please consult your cell manufacturer for details
1
2
Plating cells in 96/384-well microplates
Step1
Coat the plate with the material described in the cell
provider’s instruction manual.
Step2
Thaw, plate and culture the cells according to the cell
provider’s instruction manual.
Ca2+ dye loading to cells
Step1
Prepare the Loading buffer at +37 °C
Loading buffers
HEPES-Hank's Balanced Salt Solution
(calcium, magnesium) (pH7.4)
2 µM Fluo8-AM
0.05 % Pluronic F-127
1.25 µM Probenecid
Life Technologies #14025-092
Life Technologies #15630-056
AAT Bioquest #21083
Life Technologies # P-6866
Sigma #P8761
Loading
Step2
· Remove the culture medium
· Add 80 µL/well of +37 °C Loading buffer prepared in step 1
· Incubate the cells for 1 hour at +37 °C in 5 % CO2
Wash out
Step3
· Remove Loading buffer
· Add 100 µL/well of +37 °C HEPES-Hank's Balanced Salt Solution
NOTE: This dye loading protocol is just one example you may need to optimize it to have better performance in
your experiments.
3
FDSS Data Acquisition / Data Analysis
Instrument Set up
Step1
· Turn on the system 30 minutes before the experiment to cool the
camera and to warm the stage up to +37 °C
· Launch FDSS software
Protocol Setting
Step2
· Set the interval to 10 ms to 30 ms, and configure the sampling number
· Set the dispense parameter if dispense is necessary in the protocol.
Data acquisition and analysis
Step3
· Start assay with the configured setting protocol
· Open the measured data and analyze them
5
Measurement and Analysis examples
In-vitro toxicity study examples using iPS/ES derived cardiomyocytes
Evaluated Compound List
Compound
Arrythmia Contractability Concentration of
Compound
Description
Aspirin
Known compound as inhibitor of cyclooxygenase
which does not show cardiotoxicity in this experiment
E-4031
Known compound for hERG potassium channel
blocker
Isoproterenol
Known compound which sitimulates
cardiomyocyte influencing β-receptor
30 nM to 30 µM
10 nM to 10 µM
●
30 nM to 30 µM
●
Compound 1 Aspirin
BPM
1000
500
µM
µM
30
10
µM
µM
nM
nM
0
30
10
0
M
µM
µM
30
10
µM
µM
1
3
nM
nM
0
0
10
30
M
nM
0
30
µM
µM
30
10
µM
µM
1
3
nM
nM
0
0
10
30
M
nM
nM
0
0
0
2000
1000
20
0
3000
0
40
30 µM
4000
AMP (RFU)
60
30
10 µM
AMP
1500
80
P-P time (ms)
peak numbers/min
3 µM
P-P time
100
6
1 µM
300 nM
1
100 nM
3
30 nM
30
0M
FDSS/µCELL
Compound 2 E-4031
30 nM
BPM
20
0
2000
0
M
0
M
0
10
10
3000
1000
0
M
-5
nM
-8
-7
-6
log [Ligand,M]
0
Control
1000
30
n
10 M
0
nM
30
0
nM
1
µM
3
µM
10
µM
-20
2000
4000
10
0
40
3000
AMP (RFU)
20
60
5000
30
n
10 M
0
nM
30
0
nM
1
µM
3
µM
10
µM
40
10 µM
AMP
4000
P-P time (ms)
peak numbers/min
peak numbers/min
60
3 µM
P-P time
80
80
1 µM
300 nM
30
n
10 M
0
nM
30
0
nM
1
µM
3
µM
10
µM
IC50 from BPM
100 nM
nM
10 nM
nM
0M
Compound 3 Isoproterenol
BPM
AMP (RFU)
1000
500
4000
3000
2000
1000
µM
µM
10
30
µM
µM
1
10
0
n
30 M
0
nM
nM
µM
µM
10
30
µM
µM
1
3
10
0
n
30 M
0
nM
M
0
nM
0
M
P-P time (ms)
µM
µM
30
10
µM
-4
µM
-5
5000
0
1
-7
-6
log [Ligand,M]
0
3
Control
20
n
10 M
0
nM
30
0
nM
50
40
M
60
60
30
peak numbers/min
70
30 µM
AMP
1500
80
0
peak numbers/min
80
10 µM
P-P time
100
90
3 µM
0
EC50 from BPM
1 µM
300 nM
3
100 nM
30
30nM
30
0M
7
System Configuration
FDSS/µCELL
Cardiomyocyte package
Configuration details
FDSS/µCELL main unit with assay stage, compound wash stage with
FDSS/µCELL main unit
fluorescence sensor unit build in.
Tip mounter
Tip mounter
High speed/sensitive sensor
EM-CCD Camera with digital Interface board, camera link cable 5 m
B Excitation Light source
Light source for Fluo-4
96 tip type dispensing unit (10 µl to 200 µl)
Dispenser Heads
384 tip type dispensing unit (1 µl to 30 µl)
Heater unit
For assay plate stage
Data Analysis Unit Package
FDSS/µCELL data analysis unit equipped with high speed data acquisition
software module and beat analysis software
Dimension / Weight (Main unit)
550 mm(W) × 1600 mm(H) × 670 mm(D) / approx. 200 kg
Dimension / Weight (Data Analysis unit)
300 mm(W) × 500 mm(H) × 500 mm(D) / approx. 20 kg
[Dimensions (Unit: mm)]
670
1600
550
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Product and software package names noted in this documentation are trademarks or registered trademarks of their respective manufacturers.
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• Information furnished by HAMAMATSU is believed to be reliable. However, no responsibility is assumed for possible inaccuracies or omissions.
Specifications and external appearance are subject to change without notice.
© 2014 Hamamatsu Photonics K.K.
HAMAMATSU PHOTONICS K.K.
www.hamamatsu.com
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