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High-throughput electrophysiology:

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Haitham Salti

on 20 June 2014

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Transcript of High-throughput electrophysiology:

- complex software needed, requiring significant expertise for analysis
There are currently many validated high throughput electrophysiology platforms.
The availability of this technology enhanced the ion-channel targeted drug discovery (e.g. cardiac & neural drugs)
More efforts are required for optimizing the platforms performance and assay reproducibility

High-throughput methods
Practical considerations
High-throughput electrophysiology:
an emerging paradigm for ion-channel
screening and physiology

manual patch clamping
- glass microelectrodes patched on the cell surface - forming a gigaseal and negative pressure results
in rupturing of the cells
- recording the ionic current by clamping of the potential

- pros: high degree of flexibility, good
success rate, extremely information-rich
- cons: very low throughput, continuous
presence of a skilled operator, labor intensive

Jessica Tschernich
Haitham Salti
Lisa Britting

- single and multi-channels systems
Automated planar array electrophysiology
-only whole cell configuration possible
-planar arrays are build in a multi-well configuration
-with integrated robotic operation for cells, solution and compound handling
-limited to mamalian stable cell lines or large-scale transiently transfected cells, like CHO or HEK-293
-rightwardshift in compound IC50 values
- high initial capital investment
- generation of high quality data, needs automated data analysis
- requirements of new cell lines:
- dissociated cells
- stably expressing transfected genes
- good patch clamp characteristics
- sufficient signal amplitude
New cell-lines provide the biggest versatility for the application in patch clamping
long time & effort consuming
- Ion channels: membrane-spanning proteins
- necessary for membrane excitability and neurotransmission
- primary molecular targets of many currently used drugs
- gold-standard: manual patch clamping (mammalian cells) or
two-electrode voltage clamp (Xenopus oocytes)
- ‘bottom-up’ configuration
- used in ion-channel drug discovery and cardiac liability profiling
- seal forming substrates either of glass, silicon nitride or plastic
- computerized application of suction and mostly planar chips
- general process: 1. filling the patch clamp substrate with electrolyte
2. add cell suspensions
3. seal onto a cell
4. break through to the whole-cell configuration
5. voltage application and add the drug

automated conventional
high-throughput platform
Flyscreen (
- 2- or 4-channel system
- inverted manual patch clamp
- using the 'Flip-Tip'-Technology
- dispensed cells in the back of the tip
- suction implus drawing the cells outside
the tip until a gigaseal is formed
- internal perfusion of compounds

(mammalian cells)
automated conventional
high-throughput platform
OpusXpress (
Molecular Devices

- 8 chambers in parallel
- fully-automated
- multiple recordings
- thirty compounds per hour in duplicates
- two-electrode voltage clamp (voltage and
current electrode)
(Xenopus oocytes)
pros: cons:
- no manual handling - depending on the device
- no electrophysiologists needed
- equal in terms of data quality
- stability of recordings

Automated Electrophysiology in cardiac & neural screening
The of use automated electrophysiology in cardiac & neural screening

Automation for cardiac screening
Regulatory agencies (US FDA, EMA, Japan) issued guidance on strategies for:
In vitro Potassium current assays (hERG potassium channel)

Automation for neural screening
Neurological diseases resulting from defects in synaptic plasticity (e.g. Alzheimer, Parkinson)

Impact of drug screening on isolated ion channels is not necessarily the same as on higher order neural networks.

Its required to scale up the assessment of synaptic function to brain preparations from animals.

Cardiac medications producing heart beat disorders as a side effect (e.g. Polymorphic ventricular arrhythmia, prolongation of QT interval)
Cardiac hERG potassium channel is responsible for such effects

Pharmaceutical industry used several assays to screen the hERG channel (e.g. optical assay, radioligand binding assay or fluorescence polarization assay):
Although good predictive potential but have several limitations

This lead to increased use of electrophysiology assays and was a key drive for improving and implementing automated electrophysilogy.
Examples: Ion works, PatchXpress, QPatch and Patchliner
In vitro brain-slice extracellular field potential recording
Uses brain slice preparation

Sample preparation requires significant manual work (long time, high technical skills required)
Selection of the potential. Slice to slice differences are the greatest source of variability
Enormous data is collected from the brain slices, but its organization and analysis is a strain

Planar-array platforms
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