Cross-site comparison ofmyocyte phase II compounds
on different iPS
cardiomyocytes based on
CardioExcyte 96 recordings

Nanion Technologies

Nanion participationin CiPA Part of the leadership team in the HTS ion channel screening subteam:
5 cardiac ion channels recorded on automated patch clamp devices(Patchliner & SyncroPatch384 PE and others) Participant in the myocyte phase II study:hiPS cardiomyocytes on impedance/EFP system: the CardioExcyte96

CiPA power tools
SyncroPatch 384/768PE.

The Patchliner.
.

The CardioExcyte 96.

Highlights
CiPA Myocyte II results:• Data collection protocols• Reported endpoints• Cross-site evaluation for two CiPA sites andthree cell types

Outlook:• CardioExcyte96 data from all sites will becompiled in a publication• If other non-core sites are interested incomparing their data to ours, please contact
us at info@nanion.deCE96

Combined Impedance/Extracellular Field Potential (EFP) assay
provides mechanistic electrophysiological endpoints in vitro

 Established SOPs forElectrophysiology

Impedance/EFP measurements
in frame of the CiPA studies
ContractilityOptogenetic
stimulation

The CardioExcyte 96•

96 wells – 96 parallel sensors•

Efficient data analysis•

Automated liquid handling robot for cellseeding, medium exchange

CE96

Replacing the Incubator
CardioExcyte 96 Incubation System•

Temperature control from RT to 60°C•

CO2 Control Range 0.1 % - 20 %•Humidity Control Range 20 % - 99 %power

CO2workflow

TemperatureControl

Gas Mixer+HumidifierCE96Long and Short-term Recordings for
Chronic or Acute Pharmacology

NSP®-96Sensor plates
allow stablerecordings
over weeks
Transparent sensor platesCardioExcyte96 experimental view of mean extracellular field potential signals recorded in stem cell derived
cardiomyocytes. Random selection of multiple wells (black squares) allows the display of the impedance
recordings on the right. Data from cell type 1

CardioExcyte96 experimental view of mean impedance beat signals recorded in stem cell derived
cardiomyocytes. Random selection of multiple wells (black squares) allows the display of the impedance
recordings on the right. Data from cell type 1

CE96

Endpoints can be monitored live, while system is recording, orcan be further processed when recording is finished, offline

Parameters available to process
Impedance data

Parameters available to process
EFP (voltage) data

CE96

Online Analysis - Parameters

CE96

Online Analysis - Parameters

Automated detection
and calculation of Field
Potential Duration.

Secondary beat detection allows quantification of numberof arrhythmic events for each recorded sweep

CE96Long-term Stability of Vehicle control and Dofetilide
60% Change Average IMP
Amplitude% Change Average EFP
Amplitude

6040200-200.1% DMSO

Dofetilide-40

200-200.1% DMSO

Dofetilide-40-60Vehicle

IMP Amplitude

IMP Amplitude-6040Dofetilide

No significant amplitude changes are present in either impedance or EFP modes when comparing addition ofvehicle vs CiPA recommended concentration of Dofetilide. Bottom graphs represent average beat rate
calculations for a representative plate treated. Data from cell type 1

CE960.0
12

3

-50.0

45

plate #

-100.0

Repolarization prolongation

100.0
80.0

20.0
0.0

-60.0
-80.0

12345

plate #-50.0

100.0

Dofetilide

40.0

-40.0

0.0

-100.060.0-20.050.01234plate #

550.00.0-50.0-100.0

23plate #Vehicle

50.00.01234545

plate #

-50.0

-100.0100.0

Dofetilide

1

Beat Rate (BPM) % change

50.0

100.0Vehicle

45

Beat Rate (BPM) % change

VehicleIMP Amplitude % change

100.0

IMP Amplitude % change

Repolarization prolongation

100.0

Control Experiments: Vehicle vs Dofetilide

Dofetilide
50.00.0
1
-50.0

23

plate #

-100.0-100.0An increase in repolarization prolongation is observed post application of Dofetilide, when comparing tovehicle control. No significant changes are present in beat rate, when comparing
Dofetilide to vehicle control.

CE96High Risk: Extracellular Field Potential Pharmacology

cell type 1

cell type 2cell type 3

Extracellular field potential is prolonged post application of different high risk compounds.
Raw data examples showing one high risk compound for each cell type.

CE96

High Risk: Quinidine Pharmacology
cell type 1

cell type 1

200.0

Repolarization prolongation

Quinidine
150.0
100.0
50.0
0.0
-50.0

0.95µM3µM

9.49µM

30µM

Arrhythmic events, % of wells

Extracellular field potential isprolonged post application ofQuinidine. Compound induced
arrhythmic events, that are also
visible in impedance traces (rightpanel). Statistical comparison of
repolarization prolongation (bottomleft, cell type 1 (blue), cell type 2(green), cell type 3 (purple)).

200.0
100.0
0.0
1µM

10µM

100µM

10µM d, l Sotalol, cell type 1

700.0

Disopyramide
500.0

300.0

100.0-100.0

1µM

10µM

100µM

Vandetanib

80.0
60.0

40.0
20.00.0-20.0

0.01µM

0.1µM

1µM

10µM

Arrhythmic events, % of wells

100.00.1µM

Arrhythmic events, % of wells

Repolarization prolongation

0.1µM

Impedance (Ω)

300.0-100.0

Repolarization prolongationd, l Sotalol

Voltage (µV)

400.0Control, cell type 1

High Risk
Arrhythmic events, % of wells

Repolarization prolongation

CE96

10µM Disopyramide, cell type 1

1µM Vandetanib, cell type 1

CE96Intermediate Risk: Extracellular Field Potential Pharmacology

cell type 1

cell type 2cell type 3

Effects of different intermediate risk compounds on extracellular field potential. Raw dataexamples showing one intermediate risk compound for each cell type.

CE96

Intermediate Risk: Cisapride Pharmacology
cell type 1

cell type 1

350.0

Repolarization prolongation

300.0

CisaprideArrhythmic events, % of wells

Extracellular field potential
is prolonged post
application of Cisapride.
In impedance mode,
Cisapride induced
arrhythmic events as well,together with aprolongation of the pulsewidth.

250.0
200.0
150.0
100.0
50.0
0.0
-50.0

0.003µM

0.01µM

0.03µM

0.1µM

CE96

Intermediate Risk Compounds
Astemizole

100

Repolarization prolongation

400.0300.0
200.0

100.0
0.00.0001µM

0.001µM

0.01µM

0.1µM

Arrhythmic events, % of wells

500.0

Repolarization prolongation

Mean Beat Rate (BPM) % change

100.0

50.00.0-50.0
0.0001µM

0.001µM

030 nM

100Astemizole

150.0-100.0

500.01µM

0.1µM

300 nM

3 μM

30 μM

30 nM

100 nM-50-100.0

200.0

Ondansetron

Risperidone

500
3 nM

10 nM-50

Intermediate risk compounds prolonged the repolarization of the field potential atdifferent rates, or produced changes in beat rate.

Arrhythmic events, % of wells

600.0

Repolarization prolongation

700.0

CE96
Low Risk: Extracellular Field Potential Pharmacology
cell type 1

cell type 2cell type 3

Effects of different low risk compounds on extracellular field potential. Raw data examples
showing one low risk compound for each cell type.

CE96

Low Risk: Loratadine Pharmacology

Low Risk – cell type 3
100.0

Repolarization prolongation

60.0
40.0
20.0
0.0
-20.0

0.00095µM

0.003µM

-40.0
-60.0

-80.0

0.0094µM

0.03µM

Arrhythmic events, % of wells

80.0

Loratadine-100.0No significant extracellular field potential changes
are observed post application of Loratadine.

Low Risk: Tamoxifen Pharmacology

CE96cell type 1

cell type 1

Repolarization prolongation

-5.0
-10.0
-15.0
-20.0
-25.0
-30.0
-35.0

-40.0
-45.0

Tamoxifen

Arrhythmic events, % of wells

0.0

Extracellular fieldpotential is shortened
post application ofTamoxifen. In
impedance mode,
Tamoxifen produced ashortening the pulse
width (bottom rightgraph).

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0.0
-20.0
-40.0
-60.0
-80.0

Verapamil-100.0

0.01µM

0.1µM0.0-20.0
-40.0
-60.0

-80.0

-100.0Diltiazem

1µM

10µM

Arrhythmic events, % of wells

Repolarization prolongation

20.00.1µM Verapamil

Control, cell type 2

0.1µM Diltiazem

Impedance (Ω)

1µM

Voltage (µV)

Repolarization prolongation

0.1µM

Arrhythmic events, % of wells

0.001µM 0.01µM

Impedance (Ω)

20.0Control, cell type 2
Voltage (µV)

Low Risk compounds

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PE

Outlook: Optical Stimulation with CardioExcyte 96 SOL

Unique CardioExcyte 96 feature:
Impedance and EFP recordings from the same cells.

PE stimulation allows for an exact overlay of theOpticalsignal, providing in-depth mechanistic understandingof the contractility and electrophysiology of iPS CM.

CardioExcyte96 SOL• for in vitro optogenetic assays• 96 high power LEDs - each well is stimulated by anindividual LED• Software controlled adjustment of stimulus intensity,
pulse length and pace rate

Timed stimuli allow parallelized IMPand EFP mean beat overlays

Special thank you to the stem
cell providers for the great cells!