A novel approach to detect electrogenic transporter activity in intact cells applied
to investigate iPSC derived cardiomyocytes and neurons
Maria Barthmes1, Riccardo Rizzetto2, Andre Bazzone1, Anna Mondini2, Jean-Francois Rolland2, Niels Fertig1, Michael George1, Andrea Brüggemann1
1Nanion Technologies, Munich, Germany; 2Axxam SpA., Bresso, Italy

Measuring capacitive currents enables
resolution of small transporter currents

Establishing a new procedure to use
intact cells as a new sample typeThe measurement of capacitive induced currents enables the resolution of verysmall biological currents since it allows accumulation of the signal generated bymultiple cells

The use of purified plasma membrane vesicles (~200 nm) or artificial liposomes isestablished. We developed a protocol to use whole eukaryotic cells instead.

Validation of the method using
a CHO-SGLT1 cell line

Measurement Principle

Measurement Principle

Pharmacology comparison

Amplitude comparison

Purified membrane• Requires high amount of cells• Time and effort for purification• Very high reproducibility
Intact cells• Few cells are sufficient• Native environment• Daily cell culture necessary

For a capacitive readout (“SSM-based electrophysiology”) purified plasma
membrane or artificial liposomes are immobilized on a gold sensor. Charging andun-charging of the vesicles caused by electrogenic transport can be detected.

Validation of the method using a HEK-NCX1
cell line

Influence of cell age

ProtocolSample impermeability

Sensor Thiolozation
Establish SSM using 3 µl Lipid in decane
Add 100 µl buffer

Signal Stability

Detach cells
Centrifuge cells 5 min at 1200 rpm
Dilute to final concentration of 2500 cells / µl
Add 20 µl of cell suspension
Centrifuge sensor at 3000 rpm for 15 min

To trigger transport activity a substrate is flushed in and out of the sensor well witha fast fluidic system. It is possible to apply one or two different substrate gradients.

• After optimization it was possible to immobilize whole cells to the sensor• Success rate when using whole cells is decreased by 50% and the current
amplitude is scattering when comparing different sensors.

• An existing NCX assay was successfullytransferred using whole cells• Sensors with intact cells and membrane
fragments have comparable
pharmacological properties• The time after seeding of the cells did nothave a significant influence.
• Enclosed salts cannot be washed out of
the sensors with whole cells, indicating thatthe cells are undamaged.

• An existing assay for themeasurement of SGLT1 activity
was successfully transferred to
the new whole cell approach.
• However signal amplitudes aresmaller and• less stable compared topurified plasma membrane of
the same cell line.

Capacitive current measurement delivers functional evidence fortransporters in iPSC derived neurons and cardiomyocytes
Conclusion

Application: Evidence for NCX in iPSC derived cardiomyocytes
Comparison NCX1 in HEK and in cardiomyocytes

The use of whole cells enables the investigation of
expensive or rare cell types due to much lower
material consumption, enabling completely new
applications.
→ Primary cell cultures can be functionally tested fortransporter activity→ Transporter function can be investigated in a nativeenvironment close to physiological conditions
Using whole cells as a sample for SSM based
electrophysiology delivers smaller and lessreproducible signals compared to membrane
preparations and results in a significantly lower
success rate.
→ The use of purified membrane fragments is still thegold standard when it comes to investigation ofeukaryotic transporters in cell lines→ Using intact cells can serve as a quick method for
proof-of-principle experiments or pre-tests
By applying the newly developed method it waspossible to collect functional evidence for the
expression of several transporters in iPSC derived cells.
→ iPSC derived cardiomyocytes show NCX activity
with high amplitude which has an influence on thecells beating behavior when inhibited→ iPSC derived neurons show glutamate and GABA
transport activity→ This supports the suitability of these cells as models
for human physiology

Adherend and detached iPSC
derived cardiomyocytes

Impedance measurement: Influence of
NCX inhibition on beating rate

Application: Evidence for GABA and Glutamate transport in iPSC
derived Neurons
Adherend and detached iPSC
derived neurons (iCell Glutaneurons)

EAAT3 substrate and inhibitor assay in CHO cells

EAAT3 substrate and inhibitor assay in CHO cells

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Watanabe, et al., J. Pharmacol.Sci.102, 7 – 16 (2006)• The developed method
enabled the measurementof NCX activity in iPSC
derived cardiomyocytes• iPSC derived
cardiomyocytes show NCXspecific activity with a highamplitude• Properties of NCX incardiomyocytes was similar
to NCX1 activity inoverexpressing HEK cells• Inhibition of NCX using
impedance measurement
induced a shift in thebeating rate

IC50 SURFE2R

IC50 Literature

HIP-B

1.18 µM

10 µM (VC)

MPDC

5.1µM

5 µM (uptake, radio)

ACBD

14 µM

30µSkyblue

8.5 µM

0.8µM

PDC

0. 92 µm

1.5 µM

L-TBOA

0.29 µm

2µM

Glutamate induced currents in iCell Glutaneurons• The developed method enables thefunctional investigation of transporter
activities in iPSC derived neurons• Assays for activity measurement of EAATsand GAT were validated and applied tothe neurons• Functional expression of glutamate andGABA transporters was demonstrated inthese cells for the first time

GABA induced currents in neurons