Patch clamp is used to study individual ion channels in cells, but its manual application is time-consuming and complicated. A unique approach has been developed that enables the patch clamp experiment to be performed inside a glass pipette.
Ion channels are a drug target class of increasing importance. During the past decade, many channel proteins have been linked to diseases and pharmacological side effects. Examples include the sodium channel SCN9a playing a unique role in peripheral pain transmission and the hERG cardiac pacemaker channel responsible for deadly toxicity.
The major hurdle to developing drugs for ion channels consists of the complicated procedures required to study their function. In contrast to enzymes and G protein-coupled receptors, these proteins need to be examined in intact cells requiring physiological membrane orientation, millisecond voltage control and precise manipulation of chemical composition on both membrane sides. Tiny currents must be measured and are prone to artifacts.
Patch clamp is the only method providing the aforementioned prerequisites. However, its traditional manual application is slow and requires artistic micro-manipulation. In the last five years, several approaches have been made to automate and simplify this method. While increasing screening throughput, automated methods also face a number of restrictions.
Most automated devices perform the patch clamp experiment in a compartment resembling a planar chip with a hole drilled into it. These approaches face a variety of problems - some dependent on the chip material used and some intrinsic to the planar set-up.
Plastic materials used in planar chips show undesirable nonspecific binding especially of lipophilic drugs to the container wall. Also, ten thousands of cells must be added to the patch chamber in order to achieve a high probability of one single cell sealing the patch clamp aperture. The cells in excess provide a formidable drug sink.
The design of planar patch chambers severely limits solution exchange rates. Shear forces constitute a high risk of losing the measured cell when rapid solution exchange is attempted, leading to unacceptably low experimental success rates and high consumable costs.
flyion has developed a unique approach to perform the patch clamp experiment inside a regular glass pipette. This Flip-the-Tip method obtains a true and stable gigaseal and whole-cell preparation by simply injecting suspended cells into a pipette and towards its tip. The gigaseal inside the pipette is extremely durable. Tapping against the pipette, flushing the pipette with saline, or even moving the pipette does not break the seal. This method is ideally suited for automation and was implemented in the fully automated Flyscreen robot and the semi-automated PatchBox.
By introducing a novel patch pipette with a bowl-shaped tip - the ChipTip - the method was further improved. The ChipTip consists entirely of glass. Small volumes down to 1µl are sufficient to completely flush the extracellular compartment without detectable potency shifts due to non-specific binding. Only 30-50 cells are injected. As soon as a cell seals the pipette, the remaining cells are flushed out of the microchamber, minimising the presence of lipid membranes and drug binding proteins in the vicinity of the recorded cell.
Rotational symmetry of the ChipTip guides the drug applying capillary to the orifice of the tip and to within 200 microns of the measured cell enabling complete solution exchange in less than 50ms. This allows mimicking ligand concentration kinetics observed in natural synapses and enables the analysis of even rapidly desensitising ligand-gated channels. The flexibility to tailor the pipettes' geometry and aperture diameters facilitates the study of small cells, subcellular organelles and even bacteria.
flyion GmbH is a spin-off from the Institute of Physiology, University of Tubingen, Germany. The company was founded in 2001 as a limited liability company by Dr Albrecht Lepple-Wienhues and Dr Klaus Ferlinz.
A. Conventional patch clamp: a single cell forms a gigaseal between glass pipette and cell membrane by gentle suction.
B. Flip-the-Tip: cells are injected into a glass micropipette and sucked towards the tip producing a gigaseal inside the pipette.
C, D, E. A hollow Quartz capillary with 170µm outer diameter is inserted and centred into a ChipTip. Video frames taken 100ms apart show the laminar flow of an injected liquid towards the cell-containing tip.
