Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
If you want to understand a neuron -- or the ion channels that make it tick -- you need to measure its electrical activity. That process is called electrophysiology, and it’s traditionally accomplished using a technique called patch clamping.
Patch clamping essentially involves wiring up electrodes to the membrane of an electrically active cell and monitoring ion flow (that is, current) across the membrane as different drugs or interventions are applied. For instance, researchers looking for inhibitors of a particular ligand-gated ion channel might clamp Chinese hamster ovary (CHO) cells expressing that particular channel and bathe the cells in its cognate ligand, producing a signal. Then, potential inhibitors are added to determine if they disrupt the channel activity. In this way, researchers can understand the electrical characteristics of specific cell types and also do a little drug discovery.
The problem, according to Ali Yehia, director of IonFlux product and business development at Fluxion Biosciences, is throughput. Manual patch clamping is exceptionally laborious. Patch clamping traditionally involves micromanipulating cells under a microscope, using the manipulator to wrangle a single cell into position. Then a probe—basically a drawn-out glass pipette containing an electrode—is attached to the cell and sealed there by suction. Using feedback loops, the operator can record the cell’s baseline activity, or apply different electrical profiles, depending on the nature of the channels under study and how the experiment is run.
By this process, a single, highly trained researcher might clamp 10 or a dozen cells in a shift and consider that a rousing success. But that’s far too slow for drug discovery. “Having regular patch clampers doing this experiment is going to be slower than snails going through goo,” Yehia says. “So it’s not really a useful tool for screening.”
In recent years, developers have released tools to simplify and automate the process of patch clamping. Tedious manual patch-clamp rigs have given way to automated “planar patch-clamp” systems with sufficient throughput to play a role in the drug-development process. Instead of a micromanipulator and manually positioned electrodes, planar patch clamps query cells by sucking them onto tiny holes in the bottom of custom microtiter plates. Several such systems are commercially available, priced mostly for drug-discovery labs. Here, we take a look at six of them.
Sophion
Copenhagen, Denmark-based Sophion offers two automated planar patch-clamp systems, the QPatch and the Qube. First launched in 2005, the QPatch can perform from 8 to 48 simultaneous recordings from either a single cell or 10 averaged cells per site. The Qube, released in 2013, is a high-throughput platform based on a 384-well plate format; running four plates per hour, the Qube can collect more than 10,000 data points in an eight-hour shift.
According to Morten Sunesen, the company’s vice president of global sales and support, the ability to average signals from multiple cells has both benefits and disadvantages. On one hand, researchers collect more data and can hedge against unsuccessful patching or poorly expressing cells by testing more cells at once. However each individual cell cannot be controlled as accurately as a single cell might be. “Single-hole [experiments] offers superior voltage control,” he says.
Sophion’s microfluidics-based consumable is made of glass, offering so-called “gigaseal” performance. “When you make a seal, it’s like electrical insulation,” Sunesen says. A gigaseal connection has gigaohm resistance, meaning essentially a perfect seal between electrode and membrane, and lower noise. “Gigaseal has to do with voltage control and hence the quality of your recording,” Sunesen explains. The glass consumable is also less likely to absorb hydrophobic drug candidates, he says, leading to more accurate test results.
With microfluidic channels and precisely controlled pressure controllers, the QPatch is capable of querying voltage-gated, ligand-gated, temperature-sensitive and mechanosensitive channels, according to Sunesen—the latter stimulated by applying pressure pulses in the microfluidic channels.
Molecular Devices/Fluxion Biosciences
Molecular Devices® offers three “e-phys” platforms, the IonWorks Barracuda®, the IonFlux™ systems (developed by Fluxion and available in both 16- and 64-channel configurations) and the PatchXpress 7000A system. These range from just over $100,000 for the 16-channel IonFlux to about a half-million dollars for their other platforms.
According to James Costantin, product marketing manager for automated electrophysiology products at Molecular Devices, each of these platforms fills a different niche in the drug-development pipeline. At the top of the “funnel” is a fluorescence-based, high-throughput screening system like the Molecular Devices FLIPR high-throughput screening system, a non-e-phys platform capable of testing million-compound libraries for pennies per well. Just below that would be the IonWorks Barracuda, Costantin says, which can analyze several hundred thousand compounds for “tens of cents per well.” The PatchXpress can yield about 200 datapoints per eight-hour day for a few dollars per well. Finally, there’s the 64-channel IonFlux HT system. (The IonFlux 16, Costantin says, is intended more for academic clients than drug-discovery firms. “A lot of academic labs are buying it because it speeds the process” of patch clamp analysis, Yehia says.)
Of the three systems, Costantin says, the PatchXpress best mimics manual patch clamping, offering data of similar quality, sophisticated voltage protocols and gigaseal resistances. (IonFlux also offers a gigaseal consumable option, but the Barracuda does not, offering resistance in the megaohm range. But, says Costantin, that doesn't mean the platform cannot perform high-quality e-phys studies. "You need good hardware and good fluidics as well," he says, noting Barracuda features "a unique flow-through design” that allows it to work with rapidly desensitizing ligand-gated channels.) The PatchXpress system also performs only single-cell recording, similar to traditional setups. In contrast, the IonWorks Barracuda and IonFlux systems can perform either single-cell or population-based analyses (64 or 20 cells at once, respectively).
Whereas PatchXpress and the IonWorks Barracuda use liquid-handling robots to deliver reagents and cells, IonFlux runs entirely on microfluidics, making the system smaller and less expensive. “What we have done at IonFlux is to get rid of the robotics,” says Yehia. That, in turn, makes the system faster; instead of 45 minutes per experiment, the IonFlux system runs in as little as 20 minutes. And, Yehia adds, because of the design of the system’s microfluidic circuits, the IonFlux is capable of complete liquid turnover in about 100 milliseconds in population mode.
Nanion Technologies
Munich, Germany-based Nanion Technologies released in 2013 the SyncroPatch 384PE, capable of up to 20,000 datapoints per day, according to marketing director and senior scientist Cecilia Farre. (A 96-well version is also available.)
With two 384-well plate consumables running in parallel, the system can analyze 768 cells simultaneously with gigaohm seals, Farre says, and in 15 minutes “you can have several hundred dose-response analyses. I couldn’t even dream of that when I did my Ph.D.”
According to Farre, the SyncroPatch 384PE is unique in that it is designed to be integrated into a high-throughput robotics “highway.” She explains, “It has an open structure so that it can be served by other robots adding compound plates, recording plates, adding cells to the plate hotel, and it has a barcode scanner for this purpose. That is a truly and fully automated process.”
Such tools will surely boost the number of ion channel-targeted drugs to reach the market in the years ahead. Still, manual patch-clamp systems are not going away, says Sunesen. “We estimate there are five to six thousand manual patch-clamp rigs out there,” he says. “Automated systems can remove some of the trivial work, but there are applications" -- such as work with tissue slices, two-electrode physiology studies and monitoring cell-cell interactions, for instance -- "where automated patch clamps cannot take over.”
Bottom line: Planar patch clamping is on the rise. But though it takes time to learn manual patch clamping, it won't be time wasted.