Automated patch clamp

Automated patch clamping is beginning to replace manual patch clamping as a method to measure the electrical activity of individual cells (biology). Different techniques are used to automate patch clamp recordings from cells in cell culture and in vivo. This work has been ongoing since the late 1990s by research labs and companies trying to reduce its complexity and cost of patch clamping manually. Patch clamping for a long time was considered an art form and is still very time consuming and tedious, especially in vivo. The automation techniques try to reduce user error and variability in obtaining quality electrophysiology recordings from single cells.

Manual patch clamp

Animation showing the gigasealing process. The pipette approaches the cell and a plume of liquid flowing out of the pipette makes a small dimple on the surface of the cell. When the resistance has increased enough, a small amount of suction is applied to the pipette which draws the cell membrane into contact with the pipette tip. This creates the gigaohmn seal characteristic of a patch clamp recording.

See also: Patch clamp

The traditional manual method to patch clamp using glass pipettes was developed by Erwin Neher and Bert Sakmann and required a highly skilled technician. The technician would position the glass pipette near a cell and apply the appropriate suction to create an electrical seal between the pipette and the cell membrane. This seal ensures a quality recording by preventing any current from leaking out between the tip of the pipette and the cell membrane. This seal is made when the membrane of the cell chemically binds with the tip of the pipette so that the inside of the pipette is only connected to the cytoplasm of the cell. This membrane-glass connection or seal is called a "gigaseal".

The technician traditionally used their mouth to provide the precise pressures required to seal it to the cell. In addition to controlling the pressure, the technician must also position the pipette at precisely the correct distance from the cell so that the membrane will seal with it. Using a micromanipulator, the pipette is moved towards the cell until the technician sees a change in the electrical resistance between the fluid inside of the pipette and the surrounding fluid (see animation). This typically requires 3–12 months of training before a technician is able to reliably record from cells. The technician is essentially performing a balancing act trying to watch and manipulate several systems simultaneously (motion, pressure, and electrical signals). Unless each portion of the process is performed accurately and with the right timing, the seal will not be formed properly and the technician will have to replace the pipette and start over.

These challenges reduce the number of recordings a technician can obtain, and significantly increases the cost. Automation seeks to reduce the time, complexity and cost of manual patch clamping.

Automation systems

The automation technique changes depending on the surrounding environment of the cells. For cells in vivo, this typically means that the cells are in the brain and surrounded by other cells. This environment also contains blood vessels, dendrites, axons, and glial cells which make it harder to form a gigaseal by clogging the 1-2μm diameter pipette tip. Here, the precise pressure and position control at the pipette tip plays a big role in preventing clogging and detecting whether a cell is near the tip of the pipette as discussed above.

Cells in vitro can be suspended in a fluid, adhered to a culture dish, or be part of a piece of tissue that has been removed from the animal. These environments typically don't have to compensate for motion of the tissue due to the heartbeat or breathing of an animal. In the case of cells in suspension, the pipette is completely replaced with a microchip with holes that can create gigaseals and measure the electrical activity. Clogging is also less of an issue for cells or tissue in culture dishes because the cells and pipette can be seen through a microscope which helps the technician avoid everything but the cell of interest.

However, the automation systems all have to perform several tasks in common. They must position the cell next to tip of a pipette or some other device with a 1-2μm hole, control the pressure at the hole, and control the voltage inside the cell.

In vivo

One example of in vivo patch clamping was shown by Kodandaramaiah, et al.. In this case the pressure control consisted of a set of electronic valves and electronic pressure regulators to provide three pressures that previously provided by a technician (high pressure 800-1000mbar, low pressure 20-30mbar, and a small vacuum 15-150mbar). 3 electronic valves switched between the three pressures and atmospheric pressure. The high pressure was used to prevent clogging the pipette, the low pressure was used when searching for cells, and the vacuum was used to help the giasealing process. These were all controlled by a computer which switched the pressures as the resistance at the tip of the pipette changed.

The manula position control in this case was replaced by a a computer controlled piezoelectric micromanipulator that moved the pipette in discrete 2-3μm steps into the tissue until it made contact with a cell. This precision control is much more accurate and repeatable than manual positioning and doesn't require an operator.

The computer also calculates and tracks the change in the electrical resistance as the pipette makes contact with the cell. It sends a a voltage signal in the form of a square wave down the pipette which either exits the end of the pipette or is blocked by the cell membrane. When the membrane blocks it, the computer stops the motion of the pipette and applies suction to form the gigaseal. This portion of the automation replaces the decision making task the technician has to perform, and unlike a technician, the computer can perform this task tirelessly and with greater precision.

All of these steps are performed in the same logical sequence as manual patch clamping but doesn't require extensive training to perform and is completely controlled by the comptuer. This reduces the expense required to obtain patch clamp recordings and increased the repeatability and robustness of recording in the living brain.

In suspension

Schematic of a patch clamp system using a droplet suspension culture and gravity to position the cells above the pipette. Suction inside the pipette draws the cells to the tip of the pipette which then forms the gigaseal.

A schematic of a patch clamp chip showing a gigaseal, whole cell recording configuration, and the ion channel and whole cell currents.

Many types of systems have been developed for patch clamping cells in suspension cultures. Typically these use microchips with tiny (1-2μm) holes in a plate instead of pipettes to create the gigaseal and record from single cells. Patch chips were developed in the early 2000s as a result of the improvement of microfabrication technologies developed by the semiconductor industry. Chips are typically made from silicon, glass, PDMS, polymide.

Another system uses a traditional patch pipette and a droplet suspension culture to obtain patch clamp recordings (see figure). This has the added benefit of using traditional pipette fabrication systems that heat a glass capillary and pull it lengthwise to created the tapered tip used in patch clamping. The patch chip systems are usually more complex and expensive but have the added benefit of parallel and hands-free operation.

Since neurons don't typically grow in suspension cultures, however, other cell types are typically recorded from. In some cases, these other cell types can transfected with genes that create membrane ion channel. This means that a cell that normally doesn't have electrical activity can grow ion channels in its membrane that will generate ionic currents. Because the cells are dissociated from one another in a suspension culture, the ionic currents in that single cell can be measured in detail. This allows the scientist to study ion channel behavior in a more controlled environment without input currents from other cells as typically seen in a neural network. This is particularly useful in drug screening studies where the target is a specific protein. Because handling cells in suspension is much easier than handling cells in culture or in vivo, patch clamp recordings can be obtained much faster and more reliably. This increases the throughput of these systems and makes screening thousands of compounds possible.

The drawback to suspension culture patch clamping is that the cells or ion channels are typically not in their natural environment. If a scientist is interested in recordings from a neural network, electrical inputs from neighboring cells will not be present in this type of system. It will also change the surrounding physical and chemical environment which can significantly affect the recordings.

Useful Links

• History of high throughput patch systems
• Book chapter on planar patch clamping 'Planar Patch Clamp'

List of patch chip systems

• Port-a-Patch automated ion channel drug screening using neurons in suspension.
• QPatch
• PatchXpress
• IonWorks
• IonFlux
• SealChip

In culture

There are many in vitro methods for automated patch clamping of cultured cells or slices of brain tissue. One example includes using a patch chip like those discussed above, along with some surface treatments that cause the cultured cells to migrate to the orifices where the gigaseal is formed as they grow. By allowing the neurons to grow up in culture, they form networks spontaneously like those in the brain which is more representative of the natural tissue than isolated cells in suspension. In another case, the cells were removed from the animal and cultured on the patch chip for 2–4 hours during which time they spontaneously formed gigaseals with a polyimide and PDMS patch chip In this case, the system required no external automation equipment to form the gigaseal but is inherently automated due to the design if the device.

Another system uses a nanopipette on a precise piezo actuator stage to scan across the surface of a culture dish. As it scans across the dish, it moves the pipette up and down to maintain a constant electrical capacitance between it and the dish or cells beneath it. As it moves closer to a cell, the capacitance increases so the actuator moves the pipette away from the cell until the capacitance has decreased. This give a precise topographical map of the cells on the culture dish. After the cells have been mapped, the computer moves the pipette over the top of the desired cell and lowers it to form the gigaseal on the selected cell. This system automates the positioning portion of patch clamping in culture.

The PatchMax is another system designed to position a standard pipette over cultured cells or slices of brain tissue in a dish. The operator positions a pipette over the sample and then lets the automated software take over to lower the pipette and detect the increase in resistance of the pipette as it makes contact with the cell. At this point the program stops and the scientist creates the gigaseal manually.