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Jun 1, 2009 (Vol. 29, No. 11)

Investigating and Focusing on Ion Channels as Drug Targets

New Screening Methods Could Increase the Utility of These Abundant Proteins

  • High-Throughput Patch Clamping

    Click Image To Enlarge +
    Cellectricon’s Dynaflow platform is based on microfluidics technology that controls the solution environment around living cells.

    Since patch clamping is the gold standard for screening ion channels, there is a distinct need for high-throughput systems for primary and secondary screening. As a result, three new high-throughput systems were introduced at the conference.

    Fluxion Biosciences showed the IonFlux-HT, which looks like a traditional plate reader. Fluxion claims this system can deliver between 8,000 and 10,000 assays per day at a cost of $0.30 per data point, making it suitable for primary or secondary screening.

    “The system uses a standard 384 microplate into which cells, compounds, and reagents are loaded,” Jeff Jensen, CEO, explained. “A polymer microfluidic channel network replaces the well plate bottom, with interconnected channels forming a fluidic network. By applying pressure, cells and compounds are introduced. By applying vacuum, the cells are trapped at channel junctions and their changes in currents can be measured by the system’s integrated electrodes,” Jensen added.

    “We have tested the system with voltage-gated ion channels such as Kv2.1, hERG, and Nav1.7, and ligand-gated channels such as P2X3, TRPV1, and GABAA. We are providing the system with or without liquid-handling capability, and this means researchers can either manually pipette or integrate their own liquid handler in if they want, or purchase a fully integrated solution from Fluxion.”

    Cellectricon has gone a different route to develop its high-throughput patch clamper. The company has teamed up with The Automation Partnership and AstraZeneca to produce Dynaflow®HT for a fully automated workflow. The system contains cell and compound storage, as well as robotic liquid and plate handling, all of which allow the cells to be prepared in one place. The cells are then sucked from 96 well plates into a silica microfluidic chip. Here the cell membrane breaks and it forms a seal with the chip where the current changes are monitored.

    “This system has been custom built over the past three years, and the heart of it is the way the microfluidics work,” said Mattias Karlsson, Ph.D., CTO. “The system can currently achieve over 7,500 data points per day with competitive running costs and when we introduce our new chip later this year it will be able to generate 15,000 data points per day at a running cost of $0.10 per data point.”

    The performance of this system was validated by Dr. Dekermendjian who presented data to show that the dose-response curves were comparable to a manual patch clamp of a GlyRa ligand-gated ion channel treated with a number of different compounds. According to Dr. Karlsson, Dynaflow HT has been used with cell lines including WSS, CHO, LTK, and HEK with voltage- and ligand-gated ion channels such as GABAA and hERG, and the system is now being further tested by BioFocus DPI to screen its SoftFocus® ion channel library of compounds.

  • The Sealing Advantage

    The Fluxion and Cellectricon platforms allow the patch clamped cells to produce mega-Ohm seals, while Nanion Technologies’ SyncroPatch 96 generates giga-Ohm seals. It is this sealing that the company claims provides higher quality data recordings.

    Like Cellectricon’s system, Nanion Technologies’ is also fully automated and records from 96 cells at a time, using a glass chip to replace the glass pipette used in manual patch clamping. Cells are captured by applying suction and when they reach the opening of the chip they form a tight contact known as a gigaseal with the sensor for detection.

    “To complement our product portfolio of automated patch-clamp systems, we have developed the SyncroPatch to match the standards of industrial ion-channel screening,” explained Niels Fertig, Ph.D., CEO of Nanion Technologies. “Based on our success using glass chips, the SyncroPatch 96 obtains success rates of approximately 60 percent for stable, whole-cell recordings, with giga-Ohm seals. Giga-Ohm seals are vital for high quality recordings and for obtaining reliable compound data. At the same time, throughput is equally important for efficient ion-channel screening.

    “With the SyncroPatch 96, we want to combine the best of both worlds. We have, so far, tested cell lines including HEK293, CHO, RBL, and Jurkat with voltage- and ligand-gated ion channels such as Nav1.5, hERG, GABAA, and TRPV1. The throughput is around 5,000 data points per day at a cost per data point of $0.60, so we see this as a good secondary screening system rather than one for primary screens,” Dr. Fertig added.

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