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Columns : Oct 15, 2010 ( )
Tool to Improve Ion-Channel Screening
Instrument Seeks to Advance Ligand-Gated Aspect of Critically Important Field!--h2>
The first decade of the 21st century has delivered an extraordinary range of new technologies with relevance to early-stage ion-channel drug discovery research. The advances have been at such a pace that only ten years ago the options for evaluating compound interactions with ion channels were broadly limited to efflux and fluorescence-based high-throughput screening assays, with conventional electrophysiology reserved for more detailed evaluation of priority compounds.
Planar substrate electrophysiology techniques then started to enable compound screening against voltage-gated and slow ligand-gated ion channels. Subsequent developments in instrumentation technology have now made it possible to facilitate the application of relatively complex voltage protocols to screen up to 100,000 compounds for voltage-gated ion-channel targets.
Although voltage-gated ion-channel research has been relatively well served in terms of instrumentation, the ligand-gated ion-channel field has advanced at a slower pace. This situation has recently been resolved and a range of suitable automated electrophysiology instruments are now available, among them the Dynaflow® HT, launched by Cellectricon earlier this year. BioFocus has chosen Cellectricon as its partner of choice for ligand-gated ion-channel screening technology.
Each Dynaflow HT system combines fully automated 96-well whole-cell patch-clamp recording, with a custom-designed microfluidics-based consumable plate, which facilitates control over the length of time that ligands and compounds are perfused over cells. This instrument enables recordings to be taken with continuous voltage-clamp being applied throughout an experiment, providing control of cell membrane potential during each assay as well as enabling the creation of customized voltage step protocols for both voltage- and ligand-gated ion-channel targets.
The consumable plate has been specifically designed to be economical to manufacture and is based upon disposable chips formed from a molded polydimethylsiloxane microfluidic network bonded to a glass substrate. This molded network design brings down the cost per data point, although it does mean that seal resistance for whole-cell recordings is generally around 100 MΩ.
As a result, the system amplifiers have been specifically designed to record with mega ohm seals with automatic series resistance and capacitance compensation enhancing the recording fidelity. The plate only requires low volumes of fluids such as cells in suspension and compounds; for example, only 5 µL of a cell suspension with a density as low as 100,000 cells per mL is required to run each experiment.
In terms of functional architecture, the recording plate is composed of four individual microfluidic chips, each of which has four separate experiment units. This gives a total of 16 separate recording group experiments per plate. Each experimental unit has up to six cells that are exposed to the same concentration of ligand/compound. By taking this approach, there is a built-in facility for cell redundancy within each group of six cells, thus increasing the collection of statistically meaningful data points for each application of compound.
In addition to this, the microfluidic laminar flow technology for applying ligands (and compounds) decouples this process from liquid-handling robotics, circumventing any rate-limiting mechanical scheduling processes and avoiding potentially poorly defined compound application times.
The system operates independently for periods of at least four hours as a result of an automated cell-preparation station. This station incorporates a heated stage to maintain cells in optimal condition before dissociation and a centrifuge for removal of media before seeding plates into the recording plate. Cells are, therefore, freshly prepared for each experiment enhancing the likelihood of all cells being viable throughout a run.
In a typical eight-hour day, the system is capable of recording from around three plates per hour providing a throughput of at least 3,000 data points per day, with minimal need for an operator to be present during experiments.
The application, control, and removal of cell culture suspensions and experimental compounds are achieved by two open compound addition wells (O1 and O2), a switch valve channel (SW), and a waste well (W). Precise control over the pneumatic pressure applied to O1, SW, and O2 creates a tri-laminar flow with distinct cut-offs between each solution stream in the recording region microchannel.
During the early stages of recording, cells are loaded from well O1 and then control recording buffer perfused to obtain stable baseline recordings (Figure 1A and B). Application of vacuum to SW then disrupts the laminar streams for a time period that can be defined by the operator, and compound is applied from the O2 well (Figure 1C and D). The instrument is able to alter the compound concentration in O2 or exchange to a different compound allowing for multiple recordings from each recording group.
This method of compound application enables precise control over the duration of compound and ligand exposure to cells during recording (Figure 2), with a typical 10–90% solution exchange time of less than 30 ms. This is particularly advantageous when recording from fast ligand-gated ion channels (e.g. nAChR).
More importantly, this also produces tightly regulated reproducible assay conditions to benchmark compound activity throughout medicinal chemistry optimization studies. Table 1 shows the benchmarking of standard compound potencies against literature values, and Table 2 illustrates the reproducibility of assay data obtained using Dynaflow HT over four weeks.
While this first-generation Dynaflow HT instrument is not intended to be used as a full HTS platform, it is particularly well suited to performing smaller, focused screening campaigns against target-directed libraries and will drive the medicinal-chemistry optimization of compound activity/efficacy with greater accuracy by producing high-quality data for each compound evaluated. The initial application data here show the speed and precision necessary as well as the long-term reproducibility required for assaying against all ligand-gated ion channels including fast acting channels.
New technologies have undoubtedly revived pharmaceutical industry interest in ion-channel targets, and Dynaflow HT offers another step forward in the search for an assay format able to really open up the ion-channel field to biologists, medicinal chemists, and the industry as a whole. This renewed opportunity should ensure that the second decade of the 21st century bears the fruit of recent advances and sees an increased volume of new therapeutically relevant ion-channel modulators in development.
Andrew Southan ([email protected]) is director of ion-channel biology at BioFocus.
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