Next level toxicity screening:
From single channel to overall cell behavior
Conrad Weichbrodt1, Mohamed Kreir1, Matthias Beckler1, Alison Obergrussberger1, Ilka Rinke1, Michael George1,Sonja Stoelze-Feix1, Andrea Brüggemann 1, Niels Fertig1
1Nanion Technologies GmbH, Gabrielenstr. 9, 80636 Munich, Germany

CardioExcyte 96 Sensor Technology

Abstract

We here present the CardioExcyte 96, asystem providing a combination of Electric
Impedance Spectroscopy (EIS) as well as
Electric Field Potential (EFP) readout for anetworkofdiversecells
likeiPS
cardiomyocytes or hepatocyte-like cells
which is exemplified by toxicity effects
utilizing reference compounds such as
Dofetilide (on iPS cardiomyocytes) orParacetamol (on hepatocyte-like cells).

EFPBaseline
CE 96
Comprehensiv esafety assays

Extracellular
Field
Potential

Ion-channel
modulation:
Na +, Ca 2+, K +, hERG

electricalconduction(e.g. Long QTSyndrome)

Impedance
Contractility(e.g.
inotropiceffects)

SyncroPatch 384PE

100 nM
dofetilide

The CardioExcyte 96 combines impedance and EFP to measure effects oncontractility and ion channel modulation (left). The NSP-96 plate contains gold
electrodes embedded in each well (middle). The impedance (top) and EFP(bottom) signals are recorded from the same cell, an example is shown (right).

1. Intrinsic sensor impedance ismeasured
2

3

4

5
day

start ofmeasurement

formation of astable monolayer

synchronization

Furthermore we present the temperaturedependent activation or deactivation ofdifferent Transient Receptor Potential (TRP)
channels by means of planar patch
clamping on our HTS platforms Patchliner
and SyncroPatch 384PE as well as withhighest resolution on a single channel levelon our recently introduced Orbit mini setup.

start ofexperiment/ agent
exposureData
analysis

2. Cells

adhereformingmonolayer,Impedance
Amp

Development of the Impedance Signal

1The SyncroPatch ® 384PE is a patch clamp module that can be usedwitha currents
state-of-the-art pipetting robot, e.g. Biomek FX (BeckmanCardiac
AP andCoulter) shown here (left). Six different cardiac channels recorded
simultaneously on the SyncroPatch 384PE. Screenshot shows dataacquisition and analysis software performing recordings from HEK orCHO cells expressing the CiPA stipulated ion channels (ChanTest, aCharles River company) activated within one single experiment on theSyncroPatch 384PE.

Dofetilide induces a decrease in beat rate and EADs in
impedance (left) and EFP (right) modes.
CompoundImpedance Z

High throughput screening (HTS) assays suchas automated electrophysiological patch
clamp and impedance based assays allow
for the determination of drug effects on awhole cell level whereas artificial bilayers
provide a robust environment for theassessment of single ion channel molecules.

CardioExcyte 96 Compound Profiling
Impedance

Many lead compounds fail in the late stagesof the drug development process mainly byinflicting drug induced injury on liver, heart orotherorgans.
Therefore, devices
fordetecting possible cell toxicity in early stagesof the development process are highly
demanded.
Especially
ionchannels
represent an important class of drug targets
for in vitro pharmacological profiling.

ComplementaryExperiments
HTSof CiPA Cardiac
Panel Using theSyncroPatch 384PEa

impedance

Rat e

PW 50

EFPBRRI

AmpRat e

EffectFPDBRRI

Dofet ilide

100nM

hERG blockerCisapride

1 µM

hERG blocker

E-4031

1 nM

hERG blocker

Sot alol

1 µM

hERG blocker

Ast emizole

1 nM

hERG blocker

Quinidine

30 µM

hERG,K V,CaV,NaV blocker

Terfenadine

1 µM

hERG,NaV blocker

I soprot erenol

30 µM

β-adrenergic agonist

Nifedipine

300 nMCaV blocker

BAYK8644

1 nM

CaV act iv at or

increases

increase

3. Differential

decrease

impedancechanges detectable

Ten different compounds were tested on Cor.4U cells in
impedance and EFP modes. The results are summarized inthis table.

4. Beating pattern stabilizes
5. Pharmacology

experiment

Sager, P.T., et al., 2014.

The ion channel currents mediating the cardiac myocyte action
potential (AP; left). The voltage clamp protocol and examples of the 6different currents recorded on the SyncroPatch 384PE.

and data analysis
Cor.4U Cardiomyocytes, Axiogenesis AG

Conclusions

Toxicology Screening on Hepatocyte-like Cells

AP, EFP and ECG – How They Correlate• CardioExcyte 96 is a hybrid system combining impedance and EFP

A. Human ventricular action
potential 1

Besides Cardiomyocytes, MetaHeps cellsrecordings from the same cell.

that exhibit hepatocyte-like characteristics• Recordings arehave been studied with the CardioExcyte96.

on e.g. hepatocytes or cell

after 24 hours resulted in detectable cell

C. Surface electrocardiogram(ECG)

quality assessment.

while• The CardioExcyte 96 provides

incubation with 5 mM APAP for 48 hours led

QT-prolongation = increasing
FPD

anutilized for toxicology studies

the dashed lines). Of note, removal of APAPgraph)in• The CardioExcyte 96 can bewithin 24 hour of incubation (indicated byblue

temperature

measure effects of CiPA relevant compounds such as E4031.

impedance indicating severe cell damage(lightphysiological• The CardioExcyte 96 can be used in cardiac safety screening toled to a considerable decrease in overall

regeneration

atincubator or using the Environmental Chamber.

Application of 5 mM Paracetamol (APAP)

B. Extracellular electric fieldpotential (EFP) and cardiac
ion channel currents that
contribute to iPSCM potentials.

madecomplementary data to otherto further decrease in impedance (pinkassays such as APC.

graph).

1. Sherwood, L. (2012). Human Physiology, From Cells to Systems (8th [revised] ed.). Cengage Learning. ISBN 9781111577438.

The Orbit mini with Temperature Control•••••••

Including eFOUR 4 channel amplifier(Elements S.R.L., Cesena, I)
Smallest footprint, USB powered
Max. bandwidth: 100 kHz

Lipid bilayer

B.

TRPA1 on the Orbit Mini. A. Effect of temperature on TRPA1 activity; B. the open
probability (Po) versus the temperature and fitted with Boltzmann equation (EC50 wasfound at 14˚C). The Arrhenius plot of the same data resulted in a Q10 of 46(Literature: Q10 ~ 40).

Baez et al., 2014. Cur r ent Topics in Mem branes Vol. 74.

Heat Activated TRPV1••

Ag/AgClMicroelectrodeA.

Thermal Transient Receptor Potential(TRP) channels are an important class ofreceptors found widely distributed
throughout the mammalian central andperipheral nervous systems. They havebeen shown to be directly activated byheat or cold in physiologically relevanttemperature ranges but are also
activated by mechano-stimulation andvarious ligands. Understanding themechanisms of temperature activation
could lead to the discovery of novelcompounds with differing effects onligand activation and temperature
activation for the treatment of pain andother disease states, with fewer sideeffects.

Optional temperature control
Active heating and coolingTemperature range : 5-50°C
Software controlled

Nanopore / Ion channel

Cold Activated TRPA1

Thermo-sensitive TRP Channels

Introducing unique MECA chiptechnology: micro electrode cavity
array (Ionera GmbH, Freiburg, D)
Enabling recordings at highest
bandwidth with excellent RMS noise
behavior

B.

A.

C.

Heat Activated TRPV3
B.

A.

Micro cavity(Ø 10 - 150 µm)

25ºC

35°CTypical Noise figures of a bilayer on:
Orbit mini

Axon 200B amplifier

1.2 pA950 fA – 5.5 pA (10 KHz)

7.0 pA> 20 pA at (100 KHz)
(depending on the bilayer setup used)

5 pA

2s

Purified TRPV3 reconstituted into bilayers on the Orbit Mini. A. Effect oftemperature on TRPV3 activity; B. Effect of elevated temperature on the open
probability (Po). Fitting the data with the Boltzmann equation yields an EC50 of
31.67˚C. The Arrhenius plot of the same data results in a Q10 of 22.2 (Literature:Q10 ~17-56).

The effect of temperature on Purified TRPV1 reconstituted into lipid bilayers on the Orbit Mini.
A. Evoking activ ity of TRPV1 in bilayers by elevated temperature; B. The effect of elevatedtemperatures on the open probability (Po) of TRPV1; C. Corresponding Arrhenius plot of the samedata resulting in a Q10 of 72 (Literature: Q10 ~18 to >100).

Literature values of TRP channels taken from: 1. Mandadi, S., Roufogalis, B.D. 2008. ThermoTRP Channels in Nociceptors: Taking a lead from Capsaicin receptor TRPV1. Curr. Neuropharmacol. 6:21-38. 2. Gavva, N.R., Treanor, J.J, et al. 2008. Pharmacological blockade of the v anilloid receptor TRPV1 elicits marked hyperthermia in humans. Pain. 136: 202-210. 3. Damien S.K. Samways and Terrance M. Egan. Calcium dependent decrease in the single channel conductance of TRPV1. Pflugers Arch. 2011 Nov ; 462(5): 681–691.

Correlation of Results Obtained via Automated Patch Clamp and via Bilayer Recordings with Temperature control

0,60,40,20203040Temperature (°C)

5060-0,225303540455055-0,4-0,6-0,8-1

Q10 = 76-1,2-1,4Relative Current

0,8Log Normalized Current

010,90,80,70,60,50,40,30,20,100Log Normalized Current

1Relative Current

Temperature (°C)

Temperature (°C)

2030405060-0,52030

405060-1-1,5-2Q10 = 27-2,5-3-3,5Temperature (°C)
Response of TRPV1 and TRPV3 to an increasing temperature application in whole cell recordings onNanion’s Patchliner system. The T50 values were determ ined at 51.5 °C and 46.4 °C respectively. Notethat the overall conductance of the channels increases with the temperature.
Inducible TRPV1 CHO cell line kindly provided by Pfizer / TRPV3 HEK cell line kindly provided by Millipore

Thermodynamic analysis of TRPV1 relative current upon heat activation, Left:
Current of TRPV1 obtained during a temperature increase. Right: temperaturedependence presented in log(Po) versus temperature plot. The data resulted ina Q10 of 76 (lipid bilayers experiments Q10 = 72 and Literature: Q10 ~18 to >100).

Thermodynamic analysis of TRPV3 relative current upon heat activation, Left: Currentof TRPV3 obtained during a temperature increase. Right: temperature dependence
presented in log(Po) versus temperature plot. The data resulted in a Q10 of 27 (lipidbilayers experiments Q10 = 22 and Literature: Q10 ~17-56).