Population Patch Clamp Technology
The era of ion-channel screening using a direct electrophysiological assay has commenced, and several instruments are commercially available, including the IonWorks HT and PatchXpress 7000A systems from Molecular Devices.
These systems offer considerably higher throughput than conventional patch clamping, yet there is a need to match the throughput of surrogate assays, as well as to reduce the cost per compound assay. In addition, conventional patch clamping of a single cell at a time, the gold standard for studying ion channels, is labor intensive and not amenable to screening large numbers of compounds in early-stage drug discovery, says Costantin. Hence, Molecular Devices developed the IonWorks Quattro system with Population Patch Clamp (PPC) technology.
We introduced PPC a year ago, Costantin said. It is a second-generation technology that records the ensemble current from up to 64 cells in each recording chamber, resulting in nearly perfect success rates and highly consistent responses from one well to the next.
Costantin discussed the emergence of innovative assays that are only possible using the PPC technique, including mutational analysis, multiplexing, and a highly parallel screening assay for ligand-gated ion-channel targets.
Since biological variability, such as cell health and size and channel-expression levels, all contribute to reduced success rates in planar patch clamp systems, Costantin explained that IonWorks HT system had to find a way to compensate and increase the likelihood of obtaining at least one successful recording for every compound. Hence, IonWorks HT system pipettes compounds in quadruplicates. Thus, the probability of obtaining at least one recording for every compound using a cell line with 70% success rate is over 99%, Constantin stated, but at the expense of lower throughput.
To eliminate the quadruplicates, PPC records averaged ionic currents from a population of cells expressing a recombinant voltage-gated ion channel. Cells are plated into a 384-well PatchPlate PPC substrate, in which each well contains multiple recording sites.
The increased throughput of the IonWorks Quattro system is ideal for generating comprehensive pharmacological determinations for dozens of compounds in a single experiment, Constantin commented. Using 96- or 384-well compound plates, a wide variety of dose-response experiments can be designed, including any number of titrations from 3 concentrations, with or without replicates.